bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization
Authors: Dias Chloé , Ballout Nissrine , Morla Guillaume , Alileche Katia , Santiago Christophe , Guerrera Chiara Ida , Chaubet Adeline , Ausseil Jerome , Trudel Stephanie
Published: May 2024

Background

In mucopolysaccharidosis type III (MPS III), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described.

Methods

Here, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of MPS III patients (MPS III-EVs) to explore the EVs’ proteins and small RNA profiles using LC-MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following wild-type (WT) or MPS III-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy.

Results

Functional enrichment analysis for both proteomics and RNA sequencing data from MPS III-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with MPS III-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines.

Conclusions

This study shows, for the first time, that GAGs from patients with MPS III can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies.

Graphical abstract
Avian influenza virus neuraminidase stalk length and haemagglutinin glycosylation patterns reveal molecularly directed reassortment promoting the emergence of highly pathogenic clade 2.3.4.4b A (H5N1) viruses
Authors: Nataraj Ranjana , Chandra Anantika , Kesavardhana Sannula
Published: May 2024

The recently emerged panzootic clade 2.3.4.4b H5N1 influenza viruses show unprecedented extensive spread in wild birds and have been transmitted to several mammals, including humans. The virologic factors that have driven the success of the 2.3.4.4b H5N1 viruses, which has not been achieved by previous H5N1 clades, is unclear. We show that the 2.3.4.4b H5 haemagglutinin (HA) paired exclusively with full length (long stalk) N1 neuraminidase (NA) in birds and mammals, unlike previous clades of H5 viruses, which preferentially paired with N1 proteins with stalk deletions (short stalk). We found that the emergence of a 2.3.4.4b H5 HA with seven glycosylation sites was critical in driving its pairing with long stalk N1. The earlier H5 clades that paired with short stalk N1s showed a pattern of eight or more glycosylation sites. A prior shift in a glycosylation site from position 103 to 171 in the receptor binding domain of H5 HA and the subsequent S173A mutation that removed it triggered the emergence of 2.3.4.4b clade H5N1 viruses. Thus, the evolution of novel variations in the H5 HA and their preference for long stalk N1 pairing led to increased fitness and pathogenicity. These observations led us to establish and validate the hypothesis that the pairing of avian virus HA and NA subtypes is not stochastic but is rather molecularly programmed by HA glycosylation and NA stalk length, modulating fitness and emergence of novel avian influenza viruses.

GRAPHICAL ABSTRACT
Predicting Theory of Mind in children from the infant connectome
Authors: Schüler Clara , Berger Philipp , Grosse Wiesmann Charlotte
Published: May 2024

Our ability to reason about other people’s mental states, labeled Theory of Mind (ToM), is critical for successful human interaction. Despite its importance for human cognition, early predictors of individual ToM development are lacking. Here, we trained a computational model to identify whole-brain connectivity patterns predictive of joint attention, from resting-state fMRI data of 8-15-month-old infants, and tested whether the identified connectome would also predict ToM capacity later in development. First, the model significantly predicted joint attention scores in an independent infant sample. Crucially, the identified connectome did indeed predict ToM in children aged 2-5 years. The default network and its interaction with the ventral attention network formed dominant connections of the network, suggesting that the interplay of bottom-up attention and higher-order cognition paves the way for mature social cognition. These findings provide an early marker for individual differences in social cognitive development, with high potential for the early diagnosis of social cognitive disorders.
Effects of age on responses of principal cells of the mouse anteroventral cochlear nucleus in quiet and noise
Authors: Postolache Maggie , Connelly Graham Catherine J. , Burke Kali , Lauer Amanda M. , Xu-Friedman Matthew A.
Published: May 2024

Older listeners often report difficulties understanding speech in noisy environments. It is important to identify where in the auditory pathway hearing-in-noise deficits arise to develop appropriate therapies. We tested how encoding of sounds is affected by masking noise at early stages of the auditory pathway by recording responses of principal cells in the anteroventral cochlear nucleus (AVCN) of aging CBA/CaJ and C57BL/6J micein vivo. Previous work indicated that masking noise shifts the dynamic range of single auditory nerve fibers (ANFs), leading to elevated tone thresholds. We hypothesized that such threshold shifts could contribute to increased hearing-in-noise deficits with age if susceptibility to masking increased in AVCN units. We tested this by recording the responses of AVCN principal neurons to tones in the presence and absence of masking noise. Surprisingly, we found that masker-induced threshold shifts decreased with age in primary-like units and did not change in choppers. In addition, spontaneous activity decreased in primary-like and chopper units of old mice, with no change in dynamic range or tuning precision. In C57 mice, which undergo early onset hearing loss, units showed similar changes in threshold and spontaneous rate at younger ages, suggesting they were related to hearing loss and not simply aging. These findings suggest that sound information carried by AVCN principal cells remains largely unchanged with age. Therefore, hearing-in-noise deficits may result from other changes during aging, such as distorted across-channel input from the cochlea and changes in sound coding at later stages of the auditory pathway.

Significance Statement

Middle age and older listeners commonly experience hearing deficits in the presence of background noise. Central auditory areas have been implicated in hearing-in-noise deficits, but it is not known where these deficits arise. We performedin vivorecordings in mice of different ages at the first stage of the auditory pathway in the brain, the cochlear nucleus, to examine how encoding of sounds is perturbed by masking noise. We found that the responses of individual neurons remain largely intact with age, including the processing of tones in masking noise, despite previously documented structural and physiological degeneration of their auditory nerve inputs. This suggests that problems hearing in masking noise result from changes at other stages of the auditory pathway.
Cyclin O controls entry into the cell-cycle variant required for multiciliated cell differentiation
Authors: Damaa Michella Khoury , Serizay Jacques , Balagué Rémi , Boudjema Amélie-Rose , Faucourt Marion , Delgehyr Nathalie , Goh Kim Jee , Lu Hao , Tan Ee Kim , James Cameron T. , Faucon Catherine , Mitri Rana , Bracht Diana Carolin , Bingle Colin D. , Dunn Norris Ray , Arnold Sebastian J. , Zaragosi Laure-Emmanuelle , Barbry Pascal , Koszul Romain , Omran Heymut , Gil-Gόmez Gabriel , Escudier Estelle , Legendre Marie , Roy Sudipto , Spassky Nathalie , Meunier Alice
Published: May 2024

Multiciliated cells (MCC) ensure proper fluid circulation in various organs in metazoans. Their differentiation is marked by the massive ampliication of cilia-nucleating centrioles and is known to be controlled by various cell cycle components. Tn a companion study, we show that the differentiation of MCC is driven by a genuine cell-cycle variant characterized by sequential and wave-like expression of canonical and non-canonical cyclins such as Cyclin O (CCNO). Patients withCCNOmutations exhibit a subtype of Primary Ciliary Dyskinesia (PCD) designated as Reduced Generation of Multiple Motile Cilia (RGMC), yet the role of CCNO during MCC differentiation remains unclear. Here, using mice and human cellular models, single cell transcriptomics and functional studies, we show thatCenais activated during a strategic temporal window at the crossroads between the onset of MCC differentiation, the entry into the MCC cell cycle variant, and the activation of the centriole biogenesis program. We ind that the absence ofCenaleads to a block of MCC progenitor differentiation at the G1/S-like transition, just before the beginning of centriole formation. This leads to a complete lack of centrioles and cilia in mouse brain and human airway MCC. Altogether, our study identifies CCNO as a core regulator of entry into the MCC cell cycle variant and shows that the coupling of centriole biogenesis to an S-like phase, maintained in MCC, is dependent on CCNO.

One sentence summary

Cyclin O is necessary for multiciliated cells to enter their differentiation cell cycle variant and allows the massive amplification of centrioles, which serve as basal bodies for cilia nucleation.
Phenotypic variability between closely related Trichodesmium strains as a means to making the most out of heterogeneous microenvironments
Authors: Bizic Mina , Ionescu Danny , Wang Siyuan , Zhang Futing , Shaked Yeala
Published: May 2024

The cynobacteria Trichodesmium spp., play an important role in oceanic carbon and nitrogen cycles. Forming colonies that host diverse microorganisms likely enhances Trichodesmiums metabolic flexibility and resilience. Combining single-colony metagenomics and metatranscriptomics with a novel bioinformatic approach we investigated the community composition and transcriptional activity of 69 individual Trichodesmium thiebautii colonies collected from the northern Red Sea, Israel. Three Trichodesmium strains were detected in all colonies, each with a heterogenous yet distinct transcriptional-activity profile. N-fixation, superoxide dismutase, and ATPase activities were similar among colonies, while P and Fe uptake, as well as photosynthesis, were high in only few colonies. Colonies had no core microbiome with >70% dissimilarity in the associated microbial communities, yet, the abundance of some species was correlated with the activity of specific Trichodesmium strains. We propose Trichodesmium occupies heterogeneous microenvironments with which it deals through inter-strain phenotypic variability further fine-tuned through interactions with beneficial, non-fixed, heterotrophic colonizers.
Phage infection fronts trigger early sporulation and collective defense in bacterial populations
Authors: Măgălie Andreea , Marantos Anastasios , Schwartz Daniel A. , Marchi Jacopo , Lennon Jay T. , Weitz Joshua S.
Published: May 2024

I.

Bacteriophage (phage) infect, lyse, and propagate within bacterial populations. However, physiological changes in bacterial cell state can protect against infection even within genetically susceptible populations. One such example is the generation of endospores byBacillusand its relatives, characterized by a reversible state of reduced metabolic activity that protects cells against stressors including desiccation, energy limitation, antibiotics, and infection by phage. Here we tested how sporulation at the cellular scale impacts phage dynamics at population scales when propagating amongstB. subtilisin spatially structured environments. Initially, we found that plaques resulting from infection and lysis were approximately 3-fold smaller on lawns of sporulating wild-type bacteria vs. non-sporulating bacteria. Notably, plaque size was reduced due to an early termination of expanding phage plaques rather than the reduction of plaque growth speed. Microscopic imaging of the plaques revealed ‘sporulation rings’, i.e., spores enriched around plaque edges relative to phage-free regions. We developed a series of mathematical models of phage, bacteria, spore, and small molecules that recapitulate plaque dynamics and identify a putative mechanism: sporulation rings arise in response to lytic activity. In aggregate, sporulation rings inhibit phage from accessing susceptible cells even when sufficient resources are available for further infection and lysis. Together, our findings identify how dormancy can self-limit phage infections at population scales, opening new avenues to explore the entangled fates of phages and their bacterial hosts in environmental and therapeutic contexts.
Huntingtin CAG repeat size variations outside the Huntington’s disease complex: associations with depression and anxiety phenotypes and basal ganglia structure
Authors: Vater Magdalena , Rost Nicolas , Eckstein Gertrud , Sauer Susann , Tontsch Alina , Erhardt Angelika , Lucae Susanne , Brückl Tanja , Klopstock Thomas , Sämann Philipp G. , Binder Elisabeth B.
Published: May 2024

Huntington’s Disease (HD) is strongly associated with psychiatric symptoms, yet, associations between Huntingtin gene (HTT) CAG repeat size variations and psychiatric phenotypes outside the HD complex are still under-investigated. In this genetic case-control study we compared the distribution ofHTTCAG repeat sizes in predefined ranges between patients with major depressive disorder (MDD) (n=2136) and anxiety disorders (ANX) (n=493), and healthy controls (CON) (n=1566). We used regression models to study interactions between the alleles and associations with fine-granular clinical phenotypes and basal ganglia structure. HD mutations in the range of incomplete penetrance (36-39 repeats) were not overrepresented in patients. In participants older than 48 years, 13-20 repeats on bothHTTalleles were associated with a reduced ANX risk whereas a 13-20|21-26 combination was associated with an increased ANX risk. Post-hoc analyses confirmed a turning point around 21 repeats and trends in the same direction were detected for MDD. The joint patient|CON analysis of the full spectrum of allele combinations confirmed interaction effects and age-dependent allele|risk profiles. A short-by-long interaction effect and an age-dependent negative correlation of the short allele on the nucleus accumbens volume was detected, independently of the diagnostic group. In conclusion, we revealed thatHTTCAG repeat sizes of both alleles in the non-HD range modulate the susceptibility for common psychiatric disorders and basal ganglia structure in an age-dependent way, displaying that normal variation of the functionally diverse wildtype huntingtin protein may already impact brain function.
KDM2B is required for ribosome biogenesis and its depletion unequally affects mRNA translation
Authors: Anastas Vollter , Chavdoula Evangelia , La Ferlita Alessandro , Soysal Burak , Cosentini Ilaria , Nigita Giovanni , Kearse Michael G. , Tsichlis Philip N.
Published: May 2024

KDM2B is a JmjC domain lysine demethylase, which promotes cell immortalization, stem cell self-renewal and tumorigenesis. Here we employed a multi-omics strategy to address its role in ribosome biogenesis and mRNA translation. These processes are required to sustain cell proliferation, an important cancer hallmark. Contrary to earlier observations, KDM2B promotes ribosome biogenesis by stimulating the transcription of genes encoding ribosome biogenesis factors and ribosomal proteins, particularly those involved in the biogenesis of the 40S ribosomal subunits. Knockdown of KDM2B impaired the assembly of the small and large subunit processomes, as evidenced by specific defects in pre-ribosomal RNA processing. The final outcome was a decrease in the rate of ribosome assembly and in the abundance of ribosomes, and inhibition of mRNA translation. The inhibition of translation was distributed unequally among mRNAs with different features, suggesting that mRNA-embedded properties influence how mRNAs interpret ribosome abundance. This study identified a novel mechanism contributing to the regulation of translation and provided evidence for a rich biology elicited by a pathway that depends on KDM2B, and perhaps other regulators of translation.
Flexible behavioral adjustment to frustrative nonreward in anticipatory behavior, but not in consummatory behavior, requires the dorsal hippocampus
Authors: Hagen Christopher , Hoxha Megi , Chitale Saee , White Andre O. , Ogallar Pedro , Expósito Alejandro N. , Agüera Antonio D. R. , Torres Carmen , Papini Mauricio R. , Sabariego Marta
Published: May 2024

The hippocampus (HC) is recognized for its pivotal role in memory-related plasticity and facilitating adaptive behavioral responses to reward shifts. However, the nature of its involvement in the response to reward downshifts remains to be determined. To bridge this knowledge gap, we explored the HC’s function through a series of experiments in various tasks involving reward downshifts and using several neural manipulations in rats. In Experiment 1, complete excitotoxic lesions of the HC impaired choice performance in an 8-maze task after reducing the quantity of sugar pellet rewards. In Experiment 2, whereas chemogenetic inhibition of the dorsal HC left consummatory responses unaffected after a sucrose downshift, it significantly disrupted anticipatory behavior following a food-pellet reward reduction. Experiments 3-5 used peripheral lipopolysaccharide (LPS) treatment and found an increase in cytokine levels in the dorsal HC (dHC, Experiment 3), impaired anticipatory choice (Experiment 4), but no effect on consummatory behavior in two reward-downshift tasks. In Experiment 6, after a sucrose downshift, we found no evidence of increased activation in either the dorsal or ventral HC, as measured by c-Fos expression. These findings highlight the HC’s pivotal role in adaptively modulating anticipatory behavior in response to frustrative nonreward, while having no effect on adjustments of consummatory behavior. Spatial orientation, memory update, choice of reward signals of different value, and anticipatory vs. consummatory adjustments to reward downshift are discussed as potential mechanisms that could elucidate the specific effects observed from HC manipulations.
Biomolecular condensates sustain pH gradients at equilibrium driven by charge neutralisation
Authors: Ausserwöger Hannes , Scrutton Rob , Sneideris Tomas , Fischer Charlotte M. , Qian Daoyuan , de Csilléry Ella , Saar Kadi L. , Białek Alan Z. , Oeller Marc , Krainer Georg , Franzmann Titus M. , Wittmann Sina , Iglesias-Artola Juan M. , Invernizzi Gaetano , Hyman Anthony A. , Alberti Simon , Lorenzen Nikolai , Knowles Tuomas P. J.
Published: May 2024

Electrochemical gradients are essential to the functioning of cells and are typically formed across membranes using active transporters and require energy input to maintain them. Here, we show by contrast that biomolecular condensates are able to sustain significant pH gradients without any external energy input. We explore the thermodynamic driving forces that establish this gradient using a microfluidics-based droplet platform that allows us to sample in a continuous manner both the stability and composition of the condensates across a wide pH range. These results reveal that condensed biomolecular systems adjust the pH of the dense phase towards the isoelectric point (pI) of the component polypeptide chains. We demonstrate, on the basis of two representative systems, FUS and PGL3, that condensates can create both alkaline and acidic gradients with a magnitude exceeding one pH unit. Investigations of multicomponent protein/nucleic acid systems further show that heterotypic interactions can modulate condensate pH gradients. We further investigate using a bioinformatics approach the diversity of electrochemical properties of complex condensates by studying a large set of human condensate networks, showing that these span a wide range of mixture pIs and pH-response behaviours. In summary, our results reveal that protein condensation may present a fundamental physico-chemical mechanism for the effective segregation and optimisation of functional processes through changes in the emergent electrochemical microenvironment.
Wind Tunnels and Airflow-Driven Assays: Methods for Establishing the Cues and Orientation Mechanisms That Modulate Female Mosquito Attraction to Human Hosts
Authors: Cardé Ring T.
Published: May 2024

Understanding how female mosquitoes find a prospective host is crucial to developing means that can interfere with this process. Many methods are available to researchers studying cues and orientation mechanisms that modulate female mosquito attraction to hosts. Behaviors that can be monitored with these assays include activation, taking flight, upwind flight along an odor plume (optomotor anemotaxis), close approach to the stimulus (including hovering), and landing. Video recording can three-dimensionally document flight tracks and can correlate overall distribution patterns and moment-to-moment movements with odor contact and the presence of nearby cues such as a visual target. Here, we introduce mosquito host-seeking behaviors and methods to study them: wind tunnels (which allow orientation in free-flight), airflow-driven assays (using either tethered mosquitoes or small assay chambers that permit flight but also often dictate walking orientation), and still-air assays (wherein in odor concentration and spatial distribution are the orientation cues). We also describe factors that affect the assays and provide assay design considerations.
Collection, Fixation, and Antibody Staining ofDrosophilaEmbryos
Authors: Kidd Thomas , Evans Timothy
Published: May 2024

TheDrosophilaembryonic central nervous system has been used for decades as a model for understanding the genetic regulation of axon guidance and other aspects of neural development. Foundational studies using antibody staining to examine the embryonic ventral nerve cord in wild-type and mutant animals led to the discovery of evolutionarily conserved genes that regulate fundamental aspects of axon guidance, including midline crossing of axons. The development of the regular, segmentally repeating structure of axon pathways in the ventral nerve cord can illustrate basic principles of axon guidance to beginning students and can also be used by expert researchers to characterize new mutants, detect genetic interactions between known genes, and precisely quantify variations in gene function in engineered mutant lines. Here, we describe a protocol for collecting and fixingDrosophilaembryos and visualizing axon pathways in the embryonic ventral nerve cord using immunofluorescence or immunohistochemical staining methods. As embryogenesis inDrosophilatakes ∼24 h to complete, a 1-d collection yields embryos representing all stages of development from newly fertilized through ready-to-hatch larvae, allowing investigation of multiple developmental events within a single batch of collected embryos. The methods described in this protocol should be accessible to introductory laboratory courses as well as seasoned investigators in established research laboratories.
Voltage-Clamp Analysis of Synaptic Transmission at theDrosophilaLarval Neuromuscular Junction
Authors: Zhang Bing , Stewart Bryan
Published: May 2024

Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of theDrosophilabody wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a “ceiling effect,” because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells ofDrosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.
Focal Recording of Synaptic Currents from Single Boutons at theDrosophilaLarval Neuromuscular Junction
Authors: Zhang Bing , Stewart Bryan
Published: May 2024

Focal recording is an extracellular method for studying synaptic transmission at theDrosophilalarval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFPDrosophilastrain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.
Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes for Electrophysiological Recording inDrosophila
Authors: Zhang Bing , Stewart Bryan
Published: May 2024

Electrophysiological recording is a group of techniques used to record electrical field potentials generated by cells. These techniques rely on several types of electrodes, which can be manufactured in the laboratory. In intracellular recording, glass microelectrodes are used to pierce the cell membrane, and then to measure the electrical potential difference between the inside and the outside of the cell. Another technique, called loose patch or focal recording, is similar to intracellular recording but the electrode tip does not pierce into the cell membrane. Rather, the electrode tip is placed near a nerve or the postsynaptic side of the neuromuscular junction (NMJ) to record extracellular changes in local potentials. A third technique involves a suction electrode, which is used to draw part of the motor nerve into the electrode so that electrical pulses can be applied to elicit action potentials of the nerve. Suction electrodes are specifically used to evoke synaptic potentials at theDrosophilalarval NMJ. This protocol details some basic methods for manufacturing microelectrodes used for intracellular recording and two-electrode voltage-clamp and loose patch electrodes used for focal recording. In addition, a method is provided for manufacturing homemade suction electrodes used for nerve stimulation.
Imaging Olfactory Learning-Induced Plasticity in Vivo in theDrosophilaBrain
Authors: Boto Tamara , Tomchik Seth M.
Published: May 2024

In vivo imaging of brain activity inDrosophilaallows the dissection of numerous types of biologically important neuronal events. A common paradigm involves imaging neuronal Ca2+transients, often in response to sensory stimuli. These Ca2+transients correlate with neuronal spiking activity, which generates voltage-sensitive Ca2+influx. In addition, there is a range of genetically encoded reporters of membrane voltage and of other signaling molecules, such as second-messenger signaling cascade enzymes and neurotransmitters, enabling optical access to a range of cellular processes. Moreover, sophisticated gene expression systems enable access to virtually any single neuron or neuronal group in the fly brain. The in vivo imaging approach enables the study of these processes and how they change during salient sensory-driven events such as olfactory associative learning, when an animal (fly) is presented an odor (a conditioned stimulus) paired with an unconditioned stimulus (an aversive or appetitive stimulus) and forms an associative memory of this pairing. Optical access to neuronal events in the brain allows one to image learning-induced plasticity following the formation of associative memory, dissecting the mechanisms of memory formation, maintenance, and recall.
Mosaic Analysis with a Repressible Cell Marker (MARCM) Clone Generation inDrosophilaDendritic Arborization Neurons
Authors: Tann Jason Y. , Xu Fangke , Kimura Minami , Moore Adrian W.
Published: May 2024

Mosaic analysis with a repressible cell marker (MARCM) is used inDrosophilaresearch to create labeled homozygous mutant clones of cells in an otherwise heterozygous fly. It allows the study of the effect of embryonically lethal genes and the determination of cell autonomy for a mutant phenotype. When used in dendritic arborization (da) neurons with a fluorescent protein targeted to the plasma membrane, MARCM allows the identification of homozygous mutant neurons and clear imaging of the dendrite arbor in both live and fixed preparations. Previous protocols that outlined experimental procedures to create MARCM clones in da neurons used a heat shock promoter to drive Flippase (FLP) expression; such an approach requires laborious embryo collection and heat shock steps, and it creates clones in other tissues besides the da neurons. The updated protocol described here outlines the use of FLP expression driven by a sensory organ precursor promoter (SOP-FLP); it requires no embryo collection or manipulation steps and creates clones exclusively in the peripheral sensory neuron lineage.
Visualization of Apoptotic Ovarian Follicles duringAedes aegyptiMosquito Egg Maturation by Fluorescent Imaging Studies
Authors: Isoe Jun , Miesfeld Roger L. , Riehle Michael A.
Published: May 2024

In insects, oocyte resorption (oosorption) or follicular atresia is one of the key physiological processes and evolutionary strategies used to optimize reproductive fitness. Mosquitoes are ideal model organisms for studying egg maturation in arthropods, as their follicle development is initiated only following the ingestion of a blood meal, followed by a carefully orchestrated series of hormonally regulated events leading to egg maturation. A cohort of approximately 100 follicles per mosquito ovary begin developing synchronously. However, a significant fraction of follicles ultimately undergo apoptosis and oosorption, especially when available resources from the blood meal are limited. Therefore, simple, rapid, and reliable techniques to accurately evaluate follicular atresia are necessary to understand mechanisms underlying follicle development in insects. This protocol describes how to detect apoptotic follicle cells within theAedes aegyptimosquito ovaries using a commercially available fluorescent-labeled inhibitor of caspases (FLICA). Caspases are key players in animal apoptosis. In this assay, the FLICA reagent enters the intracellular compartment of follicles in dissected mosquito ovaries and covalently binds to active caspases. The bound reagent remains within the cell and its fluorescent signal can be observed by confocal microscopy. Although this method was specifically developed for visualizing apoptotic ovarian follicles duringAe. aegyptimosquito egg development, it should be applicable to other mosquito tissues that undergo caspase-mediated program cell death in a time-dependent manner.
Conducting an Analysis of Mosquito Flight Behaviors in a Wind Tunnel
Authors: Alonso San Alberto Diego , Rusch Claire , Riffell Jeffrey A.
Published: May 2024

Prior to conducting wind tunnel experiments, mosquitoes must be prepared for testing. Important factors and state-dependent processes of the mosquito—like the sex, age, infection status, reproductive status, or nutritional status—should be evaluated and motivated by questions and hypotheses one seeks to address. Other critical external factors that can impact the mosquitoes’ behavior and should be controlled for both in the colony and in the room where the wind tunnel experiments take place include the circadian rhythm, room temperature, light intensity, and relative humidity. Together, the internal and external factors, and wind tunnel design, ultimately control the behavior of the mosquito and, hence, the success of the experiments. In the present protocol, we describe methods using a standard wind tunnel design in which the fan pulls the air through the working section of the wind tunnel and the mosquito behavior is recorded by a multicamera system. Variations around the camera tracking system can be adapted according to the research questions being asked and include real-time tracking for both closed-loop and open-loop control of the stimulus environment or recording of the videos for off-line digitization and analysis. Within the working section, the sensory environment (odor, visual, wind) can be controlled to test the mosquito responses to different stimuli, and below we include different equipment and tools for modifying the stimuli the mosquito experiences during flight. Finally, the methods described here are applicable to multiple mosquito species, although the experiment parameters may need to be changed (e.g., ambient luminosity).
Examining Apical or Basolateral Protein Localization inAedes aegyptiTissues: Cross-Section Immunohistochemistry
Authors: Sajadi Farwa , Picinic Britney , Afifi Salwa , Paluzzi Jean-Paul V.
Published: May 2024

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues. Here, we describe an IHC protocol for examining apical versus basolateral protein staining through sectioning tissue samples from fixed, embedded tissues (e.g., IHC-paraffin) and adding primary antibodies against a target protein. This IHC protocol provides a guide for tissue fixation, sectioning, and staining of tissue samples.
Preservation of Field-Collected Mosquito Blood Meals
Authors: Reeves Lawrence E. , Burkett-Cadena Nathan D.
Published: May 2024

All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at −20°C or −80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.
Sorting Mosquito Larvae with a COPAS Machine
Authors: Marois Eric
Published: May 2024

Transgenic mosquitoes are widely used in mosquito research. To distinguish transgenic individuals from wild types, genes for fluorescent proteins are the most commonly used genetic markers in transgenic constructs, offering all the advantages of visual selection. Although manual selection under a fluorescence binocular microscope is perfect for the selection of first-generation transgenics, managing established fluorescent lines can be facilitated by complex object parametric analyzer and sorter (COPAS) sorting, which we describe in this protocol. COPAS sorting allows researchers to purify large mosquito larval populations containing only homozygous transgenic individuals, only heterozygotes, or a mix of homozygous, wild types, and heterozygotes in desired proportions. Sorting large populations of a single sex is also possible. Finally, especially when several transgenes of different fluorescence colors are inserted in the same docking site (a recombination site previously inserted in the mosquito genome, which can be used to insert new transgenes into the same locus), they can be maintained together in a single mosquito population to save insectarium space and labor. COPAS sorting can then be used to extract the desired genotype when needed and to readjust transgene frequencies every few generations in case drift is observed.
Selection of Affibody Molecules Using Staphylococcal Display
Authors: Löfblom John , Hjelm Linnea Charlotta , Dahlsson Leitao Charles , Ståhl Stefan , Lindberg Hanna
Published: May 2024

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display onStaphylococcus carnosus. Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface ofS. carnosuscells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries intoS. carnosusis less efficient and more complicated than forEscherichia coli.Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used forE. colican easily be adapted for use inS. carnosusif needed.
Whole-Brain Electrophysiology inDrosophiladuring Sleep and Wake
Authors: Van De Poll Matthew , van Swinderen Bruno
Published: May 2024

Sleep studies inDrosophila melanogasterrely mostly on behavioral read-outs to support molecular or circuit-level investigations in this model. Electrophysiology can provide an additional level of understanding in these studies to, for example, investigate changes in brain activity associated with sleep manipulations. In this protocol, we describe a procedure for performing multichannel local field potential (LFP) recordings in the fruit fly, with a flexible system that can be adapted to different experimental paradigms and situations. The approach uses electrodes containing multiple recording sites (16), allowing the acquisition of large amounts of neuronal activity data from a transect through the brain while flies are still able to sleep. The approach starts by tethering the fly, followed by positioning it on an air-supported ball. A multichannel silicon probe is then inserted laterally into the fly brain via one eye, allowing for recording of electrical signals from the retina through to the central brain. These recordings can be acquired under spontaneous conditions or in the presence of visual stimuli, and the minimal surgery promotes long-term recordings (e.g., overnight). Sleep and wake can be tracked using infrared cameras, which allow for the measurement of locomotive activity as well as microbehaviors such as proboscis extensions during sleep. The protocol has been optimized to promote subject survivability, which is an important factor when performing long-term (∼16-h) recordings. The approach described here uses specific recording probes, data acquisition devices, and analysis tools. Although it is expected that some of these items might need to be adapted to the equipment available in different laboratories, the overall aim is to provide an overview on how to record electrical activity across the brain of behaving (and sleeping) flies using this kind of approach and technology.
Experiments and Analysis of Mosquito Flight Behaviors in a Wind Tunnel: An Introduction
Authors: Alonso San Alberto Diego , Rusch Claire , Riffell Jeffrey A.
Published: May 2024

Mosquitoes detect and navigate to important resources, like a host, using combinations of different sensory stimuli. The relative importance of the sensory cues can change as the mosquito gets closer to their target. Other factors, both internal and external, can also influence the mosquito behavior. A mechanistic understanding of these sensory stimuli, and how they impact mosquito navigation, can now be readily studied using wind tunnels and associated computer vision systems. In this introduction, we present a behavioral paradigm using a wind tunnel for flight behavior analysis. The wind tunnel's large size with its associated cameras and software system for analysis of the mosquito flight tracks can be sophisticated and sometimes cost-prohibitive. Nevertheless, the wind tunnel's flexibility in allowing the testing of multimodal stimuli and scaling of environmental stimuli makes it possible to reproduce conditions from the field and test them in the laboratory, while also allowing the observation of natural flight kinematics.
Functional Imaging of Learning-Induced Plasticity in the Central Nervous System with Genetically Encoded Reporters inDrosophila
Authors: Boto Tamara , Tomchik Seth M.
Published: May 2024

Learning and memory allow animals to adjust their behavior based on the predictive value of their past experiences. Memories often exist in complex representations, spread across numerous cells and synapses in the brain. Studying relatively simple forms of memory provides insights into the fundamental processes that underlie multiple forms of memory. Associative learning occurs when an animal learns the relationship between two previously unrelated sensory stimuli, such as when a hungry animal learns that a particular odor is followed by a tasty reward.Drosophilais a particularly powerful model to study how this type of memory works. The fundamental principles are widely shared among animals, and there is a wide range of genetic tools available to study circuit function in flies. In addition, the olfactory structures that mediate associative learning in flies, such as the mushroom body and its associated neurons, are anatomically organized, relatively well-characterized, and readily accessible to imaging. Here, we review the olfactory anatomy and physiology of the olfactory system, describe how plasticity in the olfactory pathway mediates learning and memory, and explain the general principles underlying calcium imaging approaches.
Analysis of Axon Guidance in theDrosophilaEmbryo
Authors: Kidd Thomas , Evans Timothy
Published: May 2024

The establishment of neural connectivity is a major part of neural development. The central nervous system (CNS) midline is the most characterized axon guidance choice point, and work inDrosophilahas played a pivotal role in understanding the molecular mechanisms responsible. Axons respond to attractive cues such as Netrin via the Frazzled receptor, and repulsive cues such as Slit via Robo receptors. Both signals are expressed at the CNS midline, affect pioneer axons, and have dramatic effects on the axon scaffold as a whole. Here, we focus on previous research analyzing classic mutants in the Slit/Robo pathway, which can readily be detected with a dissecting microscope. We also discuss analyzing these mutants in a teaching lab situation. The combination of sophisticated genetics and reliable axonal markers inDrosophilaallows phenotypic analysis to be performed at the single-cell level. The elaborate architecture of neurons is very sensitive to disruption by genetic mutations, allowing the effects of novel mutations to be easily detected and assessed.
Electron-photon exchange-correlation approximation for quantum-electrodynamical density-functional theory
Authors: Lu I-Te , Ruggenthaler Michael , Tancogne-Dejean Nicolas , Latini Simone , Penz Markus , Rubio Angel
Published: May 2024

Quantum-electrodynamical density-functional theory (QEDFT) provides a promising avenue for exploring complex light-matter interactions in optical cavities for real materials. Similar to conventional density-functional theory, the Kohn-Sham formulation of QEDFT needs approximations for the generally unknown exchange-correlation functional. In addition to the usual electron-electron exchange-correlation potential, an approximation for the electron-photon exchange-correlation potential is needed. A recent electron-photon exchange functional [C. Schäfer , ], derived from the equation of motion of the nonrelativistic Pauli-Fierz Hamiltonian, shows robust performance in one-dimensional systems across weak- and strong-coupling regimes. Yet, its performance in reproducing electron densities in higher dimensions remains unexplored. Here we consider this QEDFT functional approximation from one- to three-dimensional finite systems and across weak to strong light-matter couplings. The electron-photon exchange approximation provides excellent results in the ultrastrong-coupling regime. However, to ensure accuracy also in the weak-coupling regime across higher dimensions, we introduce a computationally efficient renormalization factor for the electron-photon exchange functional, which accounts for part of the electron-photon correlation contribution. These findings extend the applicability of photon-exchange-based functionals to realistic cavity-matter systems, fostering the field of cavity QED (quantum-electrodynamics) materials engineering.

Published by the American Physical Society
2024