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.

Non-technical summary Elevated systemic haematocrit (Hct) increases cardiovascular risk, 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 gel-like layer (the 'glycocalyx' , or 'endothelial surface layer' -ESL) that modulates various biological processes, including inflammation, permeability and atherosclerosis. However, the consequences of an elevated Hct on the functional properties of this interface are incompletely understood. In a transgenic mouse (tg6) model exhibiting systemic Hct levels of about 0.85 the glycocalyx/ESL was nearly abolished. The corresponding increase in vessel diameter had only minor effects on peripheral flow resistance. This suggests that the pathological effects of elevated Hct may relate more strongly to the biological corollaries of a reduced ESL thickness and alterations of the blood-endothelium interface than to an increased flow resistance.Abstract 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.

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.

Animals often use sensory cells with opposing polarity that are separately tuned to detect increments or decrements of environmental stimuli. For example, cooling and warming cells detect temperature changes1,2; moist and dry cells detect humidity changes3,4; and ON-OFF retinal cells detect luminance changes5,6. How animals integrate the outputs of opponent cells to navigate towards favorable points along a sensory gradient from higher or lower points remains poorly understood. Here, we show how the outputs of warming cells (WCs) and cooling cells (CCs) in the Drosophila larva are combined to produce context-dependent thermotactic navigation. The newly discovered WCs and the previously described CCs7 require distinct but overlapping sets of Ionotropic Receptors to sense temperature changes: Ir68a, Ir93a, and Ir25a for WCs; Ir21a, Ir93a, and Ir25a for CCs. WCs and CCs are bidirectional opponent sensors: WCs are activated by warming and inhibited by cooling, whereas CCs are activated by cooling and inhibited by warming. Both WCs and CCs are sensitive to temperature changes from high temperatures where the larva avoids warming to low temperatures where it avoids cooling. However, at all temperatures, optogenetically-imposed fictive warming of the WCs as well as fictive cooling of the CCs evoke avoidance responses, and fictive cooling of the WCs as well as fictive warming of the CCs inhibit avoidance responses. We reconcile these sensory properties and behavioral responses by showing that the larva uses flexible cross-inhibition of the output pathways of WCs and CCs when making perceptual choices during thermotaxis. Balanced cross-inhibition near preferred temperatures suppresses any directed movement on temperature gradients. Above preferred temperatures, WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling. Below preferred temperatures, CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. Our results show how flexible cross-inhibition orchestrates a context-dependent perceptual choice during a navigational behavior.

TheDrosophilalarva 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.Drosophilalarvae 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 theGAL4/UASsystem. 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.