The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila.

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Learning and Memory in Drosophila Larvae.

The Drosophila larva has become an attractive model system for studying fundamental questions in neuroscience. Although the focus was initially on topics such as the structure of genes, mechanisms of inheritance, genetic regulation of development, and the function and physiology of ion channels, today it is often on the cellular and molecular principles of naive and learned behavior. Drosophila larvae have developed different mechanisms, often widespread in similar manifestations in the animal kingdom, to orient themselves toward olfactory, gustatory, mechanosensory, thermal, and visual stimuli to coordinate their locomotion appropriately. To adapt to changes in the environment, larvae are able to learn to categorize some of these sensory impressions as "good" or "bad." Depending on their relevance and reliability, the larva learns them and constantly updates these memories. Laboratory experiments allow us to parametrically study and describe many of these processes (e.g., olfactory appetitive and aversive memory or visual appetitive and aversive memory). Combining behavioral tests with various neurogenetic techniques allows us to thermally or optogenetically activate or inhibit individual cells during learning, memory consolidation, and memory retrieval. The molecular and genetic bases of larval learning can be analyzed by using specific mutants. The CRISPR-Cas method has established extensive new directions in this area, in addition to the already wide-ranging traditional approaches, like the GAL4/UAS system. The combination of these genetic methods with the simplicity and cost-effectiveness of the introduced behavioral assay provides a platform for discovering the fundamental mechanisms underlying learning and memory formation in the rather simple larval brain.

The Analysis of Aversive Olfactory-Taste Learning and Memory in Drosophila Larvae.

Drosophila larvae are able to associate an odor stimulus with a temporally overlapping teaching signal encoding reward or punishment. Here, we describe a standardized experimental setup that allows the analysis of larval aversive-odor-taste learning and memory. This is a Pavlovian learning experiment with a single training trial in which larvae are presented with two specific odors in succession, one odor together with salt at a high concentration that is harmful to the larva. In the subsequent test, the trained larvae then show avoidance of the salt-paired odor and spend more time near the unpaired odor. To rule out nonassociative effects (such as naive preferences for odors, exposure, or handling effects), two independent groups of larvae are reciprocally trained. Subsequently, the average of the two individual preference values is determined and quantified as a Performance Index (PI), which assigns a numerical value to the larvae's shown behavioral change.

Somatic copy number variant load in neurons of healthy controls and Alzheimer's disease patients.

The possible role of somatic copy number variations (CNVs) in Alzheimer's disease (AD) aetiology has been controversial. Although cytogenetic studies suggested increased CNV loads in AD brains, a recent single-cell whole-genome sequencing (scWGS) experiment, studying frontal cortex brain samples, found no such evidence. Here we readdressed this issue using low-coverage scWGS on pyramidal neurons dissected via both laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) across five brain regions: entorhinal cortex, temporal cortex, hippocampal CA1, hippocampal CA3, and the cerebellum. Among reliably detected somatic CNVs identified in 1301 cells obtained from the brains of 13 AD patients and 7 healthy controls, deletions were more frequent compared to duplications. Interestingly, we observed slightly higher frequencies of CNV events in cells from AD compared to similar numbers of cells from controls (4.1% vs. 1.4%, or 0.9% vs. 0.7%, using different filtering approaches), although the differences were not statistically significant. On the technical aspects, we observed that LCM-isolated cells show higher within-cell read depth variation compared to cells isolated with FACS. To reduce within-cell read depth variation, we proposed a principal component analysis-based denoising approach that significantly improves signal-to-noise ratios. Lastly, we showed that LCM-isolated neurons in AD harbour slightly more read depth variability than neurons of controls, which might be related to the reported hyperploid profiles of some AD-affected neurons.

Synchronous and opponent thermosensors use flexible cross-inhibition to orchestrate thermal homeostasis.

Body temperature homeostasis is essential and reliant upon the integration of outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these outputs are not understood. Here, we discover a set of warming cells (WCs) and show that the outputs of these WCs combine with previously described cooling cells (CCs) in a cross-inhibition computation to drive thermal homeostasis in larval Drosophila WCs and CCs detect temperature changes using overlapping combinations of ionotropic receptors: Ir68a, Ir93a, and Ir25a for WCs and Ir21a, Ir93a, and Ir25a for CCs. WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling, and CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Ambient temperature-dependent regulation of the strength of WC- and CC-mediated cross-inhibition keeps larvae near their homeostatic set point. Using neurophysiology, quantitative behavioral analysis, and connectomics, we demonstrate how flexible integration between warming and cooling pathways can orchestrate homeostatic thermoregulation.

A new method to characterize function of the Drosophila heart by means of optical flow.

The minuteness of Drosophila poses a challenge to quantify performance of its tubular heart and computer-aided analysis of its beating heart has evolved as a resilient compromise between instrumental costs and data robustness. Here, we introduce an optical flow algorithm (OFA) that continuously registers coherent movement within videos of the beating Drosophila heart and uses this information to subscribe the time course of observation with characteristic phases of cardiac contraction or relaxation. We report that the OFA combines high discriminatory power with robustness to characterize the performance of the Drosophila tubular heart using indicators from human cardiology. We provide proof of this concept using the test bed of established cardiac conditions that include the effects of ageing, knockdown of the slow repolarizing potassium channel subunit KCNQ and ras-mediated hypertrophy of the heart tube. Together, this establishes the analysis of coherent movement as a suitable indicator of qualitative changes of the heart's beating characteristics, which improves the usefulness of Drosophila as a model of cardiac diseases.

Excessive erythrocytosis compromises the blood-endothelium interface in erythropoietin-overexpressing mice.

Elevated systemic haematocrit (Hct) increases risk of cardiovascular disorders, such as stroke and myocardial infarction. One possible pathophysiological mechanism could be a disturbance of the blood-endothelium interface. It has been shown that blood interacts with the endothelial surface via a thick hydrated macromolecular layer (the 'glycocalyx', or 'endothelial surface layer'--ESL), modulating various biological processes, including inflammation, permeability and atherosclerosis. However, the consequences of elevated Hct on the functional properties of this interface are incompletely understood. Thus, we combined intravital microscopy of an erythropoietin overexpressing transgenic mouse line (tg6) with excessive erythrocytosis (Hct 0.85), microviscometric analysis of haemodynamics, and a flow simulation model to assess the effects of elevated Hct on glycocalyx/ESL thickness and flow resistance. We show that the glycocalyx/ESL is nearly abolished in tg6 mice (thickness: wild-type control: 0.52 µm; tg6: 0.13 µm; P < 0.001). However, the corresponding reduction in network flow resistance contributes <20% to the maintenance of total peripheral resistance observed in tg6 mice. This suggests that the pathological effects of elevated Hct in these mice, and possibly also in polycythaemic humans, may relate to biological corollaries of a reduced ESL thickness and the consequent alteration in the blood-endothelium interface, rather than to an increase of flow resistance.