Integration of Parallel Opposing Memories Underlies Memory Extinction.

Accurately predicting an outcome requires that animals learn supporting and conflicting evidence from sequential experience. In mammals and invertebrates, learned fear responses can be suppressed by experiencing predictive cues without punishment, a process called memory extinction. Here, we show that extinction of aversive memories in Drosophila requires specific dopaminergic neurons, which indicate that omission of punishment is remembered as a positive experience. Functional imaging revealed co-existence of intracellular calcium traces in different places in the mushroom body output neuron network for both the original aversive memory and a new appetitive extinction memory. Light and ultrastructural anatomy are consistent with parallel competing memories being combined within mushroom body output neurons that direct avoidance. Indeed, extinction-evoked plasticity in a pair of these neurons neutralizes the potentiated odor response imposed in the network by aversive learning. Therefore, flies track the accuracy of learned expectations by accumulating and integrating memories of conflicting events.

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.

Dopaminergic systems create reward seeking despite adverse consequences.

Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking1. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking2,3, the underlying reasons for this behaviour are unclear. Here we describe dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. We propose that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals.

Hungry flies tune to vinegar.

Many molecular signals that represent hunger and satiety in the body have been identified, but relatively little is known about how these factors alter the nervous system to change behavior. Root et al. (2011) report that hunger modulates the sensitivity of specific olfactory sensory neurons in Drosophila and facilitates odor-search behavior.

Van Gogh-like 2 is essential for the architectural patterning of the mammalian biliary tree.

BACKGROUND & AIMS: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFß signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct. METHODS: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning. RESULTS: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation. CONCLUSIONS: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture. IMPACT AND IMPLICATIONS: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed.

A neural circuit mechanism integrating motivational state with memory expression in Drosophila.

Behavioral expression of food-associated memory in fruit flies is constrained by satiety and promoted by hunger, suggesting an influence of motivational state. Here, we identify a neural mechanism that integrates the internal state of hunger and appetitive memory. We show that stimulation of neurons that express neuropeptide F (dNPF), an ortholog of mammalian NPY, mimics food deprivation and promotes memory performance in satiated flies. Robust appetitive memory performance requires the dNPF receptor in six dopaminergic neurons that innervate a distinct region of the mushroom bodies. Blocking these dopaminergic neurons releases memory performance in satiated flies, whereas stimulation suppresses memory performance in hungry flies. Therefore, dNPF and dopamine provide a motivational switch in the mushroom body that controls the output of appetitive memory.

Transposon expression in the Drosophila brain is driven by neighboring genes and diversifies the neural transcriptome.

Somatic transposon expression in neural tissue is commonly considered as a measure of mobilization and has therefore been linked to neuropathology and organismal individuality. We combined genome sequencing data with single-cell mRNA sequencing of the same inbred fly strain to map transposon expression in the Drosophila midbrain and found that transposon expression patterns are highly stereotyped. Every detected transposon is resident in at least one cellular gene with a matching expression pattern. Bulk RNA sequencing from fly heads of the same strain revealed that coexpression is a physical link in the form of abundant chimeric transposon-gene mRNAs. We identified 264 genes where transposons introduce cryptic splice sites into the nascent transcript and thereby significantly expand the neural transcript repertoire. Some genes exclusively produce chimeric mRNAs with transposon sequence; on average, 11.6% of the mRNAs produced from a given gene are chimeric. Conversely, most transposon-containing transcripts are chimeric, which suggests that somatic expression of these transposons is largely driven by cellular genes. We propose that chimeric mRNAs produced by alternative splicing into polymorphic transposons, rather than transposon mobilization, may contribute to functional differences between individual cells and animals.

DKK1 drives immune suppressive phenotypes in intrahepatic cholangiocarcinoma and can be targeted with anti-DKK1 therapeutic DKN-01.

BACKGROUND AND AIMS: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth. METHODS: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage-induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01. RESULTS: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease. CONCLUSION: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.

Re-evaluation of learned information in Drosophila.

Animals constantly assess the reliability of learned information to optimize their behaviour. On retrieval, consolidated long-term memory can be neutralized by extinction if the learned prediction was inaccurate. Alternatively, retrieved memory can be maintained, following a period of reconsolidation during which it is labile. Although extinction and reconsolidation provide opportunities to alleviate problematic human memories, we lack a detailed mechanistic understanding of memory updating. Here we identify neural operations underpinning the re-evaluation of memory in Drosophila. Reactivation of reward-reinforced olfactory memory can lead to either extinction or reconsolidation, depending on prediction accuracy. Each process recruits activity in specific parts of the mushroom body output network and distinct subsets of reinforcing dopaminergic neurons. Memory extinction requires output neurons with dendrites in the α and α' lobes of the mushroom body, which drive negatively reinforcing dopaminergic neurons that innervate neighbouring zones. The aversive valence of these new extinction memories neutralizes previously learned odour preference. Memory reconsolidation requires the γ2α'1 mushroom body output neurons. This pathway recruits negatively reinforcing dopaminergic neurons innervating the same compartment and re-engages positively reinforcing dopaminergic neurons to reconsolidate the original reward memory. These data establish that recurrent and hierarchical connectivity between mushroom body output neurons and dopaminergic neurons enables memory re-evaluation driven by reward-prediction error.

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.

Memory, anticipation, action - working with Troy D. Zars.

We present here our reflections on the scientific work of the late Troy D. Zars (1967 - 2018), on what it was like to work with him, and what it means to us. A common theme running through his work is that memory systems are not for replaying the past. Rather, they are forward-looking systems, providing whatever guidance past experience has to offer for anticipating the outcome of future actions. And in situations where no such guidance is available trying things out is the best option. Working with Troy was inspiring precisely because of the optimism inherent in this concept and that he himself embodied. Our reflections highlight what this means to us as his former mentors, colleagues, and mentees, respectively, and what it might mean for the future of neurogenetics.

Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains.

RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. Our simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, we introduce the use of smFISH as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain.

Layered reward signalling through octopamine and dopamine in Drosophila.

Dopamine is synonymous with reward and motivation in mammals. However, only recently has dopamine been linked to motivated behaviour and rewarding reinforcement in fruitflies. Instead, octopamine has historically been considered to be the signal for reward in insects. Here we show, using temporal control of neural function in Drosophila, that only short-term appetitive memory is reinforced by octopamine. Moreover, octopamine-dependent memory formation requires signalling through dopamine neurons. Part of the octopamine signal requires the α-adrenergic-like OAMB receptor in an identified subset of mushroom-body-targeted dopamine neurons. Octopamine triggers an increase in intracellular calcium in these dopamine neurons, and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking octopamine. Analysis of the ß-adrenergic-like OCTß2R receptor reveals that octopamine-dependent reinforcement also requires an interaction with dopamine neurons that control appetitive motivation. These data indicate that sweet taste engages a distributed octopamine signal that reinforces memory through discrete subsets of mushroom-body-targeted dopamine neurons. In addition, they reconcile previous findings with octopamine and dopamine and suggest that reinforcement systems in flies are more similar to mammals than previously thought.

Cryptochrome mediates light-dependent magnetosensitivity in Drosophila.

Although many animals use the Earth's magnetic field for orientation and navigation, the precise biophysical mechanisms underlying magnetic sensing have been elusive. One theoretical model proposes that geomagnetic fields are perceived by chemical reactions involving specialized photoreceptors. However, the specific photoreceptor involved in such magnetoreception has not been demonstrated conclusively in any animal. Here we show that the ultraviolet-A/blue-light photoreceptor cryptochrome (Cry) is necessary for light-dependent magnetosensitive responses in Drosophila melanogaster. In a binary-choice behavioural assay for magnetosensitivity, wild-type flies show significant naive and trained responses to a magnetic field under full-spectrum light ( approximately 300-700 nm) but do not respond to the field when wavelengths in the Cry-sensitive, ultraviolet-A/blue-light part of the spectrum (<420 nm) are blocked. Notably, Cry-deficient cry(0) and cry(b) flies do not show either naive or trained responses to a magnetic field under full-spectrum light. Moreover, Cry-dependent magnetosensitivity does not require a functioning circadian clock. Our work provides, to our knowledge, the first genetic evidence for a Cry-based magnetosensitive system in any animal.

Compensatory enhancement of input maintains aversive dopaminergic reinforcement in hungry Drosophila.

Hungry animals need compensatory mechanisms to maintain flexible brain function, while modulation reconfigures circuits to prioritize resource seeking. In Drosophila, hunger inhibits aversively reinforcing dopaminergic neurons (DANs) to permit the expression of food-seeking memories. Multitasking the reinforcement system for motivation potentially undermines aversive learning. We find that chronic hunger mildly enhances aversive learning and that satiated-baseline and hunger-enhanced learning require endocrine adipokinetic hormone (AKH) signaling. Circulating AKH influences aversive learning via its receptor in four neurons in the ventral brain, two of which are octopaminergic. Connectomics revealed AKH receptor-expressing neurons to be upstream of several classes of ascending neurons, many of which are presynaptic to aversively reinforcing DANs. Octopaminergic modulation of and output from at least one of these ascending pathways is required for shock- and bitter-taste-reinforced aversive learning. We propose that coordinated enhancement of input compensates for hunger-directed inhibition of aversive DANs to preserve reinforcement when required.

A TGFß-ECM-integrin signaling axis drives structural reconfiguration of the bile duct to promote polycystic liver disease.

The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-ß (TGFß) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFß signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.