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  • 1
    In: eLife, eLife Sciences Publications, Ltd, Vol. 12 ( 2023-01-03)
    Abstract: Cephalopods are set apart from other mollusks by their advanced behavioral abilities and the complexity of their nervous systems. Because of the great evolutionary distance that separates vertebrates from cephalopods, it is evident that higher cognitive features have evolved separately in these clades despite the similarities that they share. Alongside their complex behavioral abilities, cephalopods have evolved specialized cells and tissues, such as the chromatophores for camouflage or suckers to grasp prey. Despite significant progress in genome and transcriptome sequencing, the molecular identities of cell types in cephalopods remain largely unknown. We here combine single-cell transcriptomics with in situ gene expression analysis to uncover cell type diversity in the European squid Loligo vulgaris . We describe cell types that are conserved with other phyla such as neurons, muscles, or connective tissues but also cephalopod-specific cells, such as chromatophores or sucker cells. Moreover, we investigate major components of the squid nervous system including progenitor and developing cells, differentiated cells of the brain and optic lobes, as well as sensory systems of the head. Our study provides a molecular assessment for conserved and novel cell types in cephalopods and a framework for mapping the nervous system of L. vulgaris .
    Type of Medium: Online Resource
    ISSN: 2050-084X
    Language: English
    Publisher: eLife Sciences Publications, Ltd
    Publication Date: 2023
    detail.hit.zdb_id: 2687154-3
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  • 2
    Online Resource
    Online Resource
    Frontiers Media SA ; 2022
    In:  Frontiers in Physiology Vol. 13 ( 2022-9-14)
    In: Frontiers in Physiology, Frontiers Media SA, Vol. 13 ( 2022-9-14)
    Abstract: The inclusion of cephalopods in the legislation related to the use of animals for experimental purposes has been based on the precautionary principle that these animals have the capacity to experience pain, suffering, distress, and lasting harm. Recent studies have expanded this view and supported it. Handling cephalopod mollusks in research is challenging and whenever more invasive procedures are required, sedation and/or anesthesia becomes necessary. Therefore, finding adequate, safe, and effective anesthetics appears mandatory. Several substances have been considered in sedating cephalopods, in some instances applying those utilized for fish. However, species-specific variability requires more detailed studies. Despite long-lasting experience being linked to classic studies on squid giant axons, evidence of action on putative anesthetic substances is scarce for Loligo vulgaris and particularly for their embryos. The aim of the current study was to evaluate effects elicited by immersion of squid embryos in anesthetic solutions and examine whether these forms display a similar reaction to anesthetics as adults do. Different concentrations of ethanol (EtOH; 2, 2.5, and 3%) and magnesium chloride (MgCl 2 ; 1, 1.5, and 1.8%) were tested by adopting a set of indicators aimed at exploring the physiological responses of squid embryos. Forty-two embryos of the common squid Loligo vulgaris (stages 27–28) were assigned to three conditions (EtOH, MgCl 2, and controls) and video recorded for 15 min (5 min before, 5 min during, and 5 min after immersion in the anesthetic solutions). In each group, the heart rate, respiratory rate, buoyancy, chromatophore activity, and tentacles/arms responses were assessed to evaluate the embryos' vitality and responsiveness to stimulation. Both substances provoked a decrease in heart and respiratory rates and inhibited buoyancy, chromatophores, and tentacles/arms responses; no adverse effects were observed. EtOH had a faster onset of action and faster recovery than MgCl 2 , being potentially more adequate as an anesthetic for shorter procedures. Even though MgCl 2 caused a longer muscle relaxation, the reversibility was not confirmed for the 1.8% concentration; however, lower concentrations triggered similar results as the ones obtained with the highest EtOH concentrations. We have shown that the late developmental stages of Loligo vulgaris embryos could represent a good model to evaluate anesthetics for cephalopods since they can display similar reactions to anesthetics as adults animals do.
    Type of Medium: Online Resource
    ISSN: 1664-042X
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2564217-0
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  • 3
    In: Biology Open, The Company of Biologists, Vol. 3, No. 12 ( 2014-12-15), p. 1252-1261
    Abstract: Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.
    Type of Medium: Online Resource
    ISSN: 2046-6390
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2014
    detail.hit.zdb_id: 2632264-X
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  • 4
    Online Resource
    Online Resource
    Society for Neuroscience ; 2020
    In:  eneuro Vol. 7, No. 2 ( 2020-03), p. ENEURO.0510-19.2020-
    In: eneuro, Society for Neuroscience, Vol. 7, No. 2 ( 2020-03), p. ENEURO.0510-19.2020-
    Abstract: Feeding, a critical behavior for survival, consists of a complex series of behavioral steps. In Drosophila larvae, the initial steps of feeding are food choice, during which the quality of a potential food source is judged, and ingestion, during which the selected food source is ingested into the digestive tract. It remains unclear whether these steps employ different mechanisms of neural perception. Here, we provide insight into the two initial steps of feeding in Drosophila larva. We find that substrate choice and ingestion are determined by independent circuits at the cellular level. First, we took 22 candidate bitter compounds and examined their influence on choice preference and ingestion behavior. Interestingly, certain bitter tastants caused different responses in choice and ingestion, suggesting distinct mechanisms of perception. We further provide evidence that certain gustatory receptor neurons (GRNs) in the external terminal organ (TO) are involved in determining choice preference, and a pair of larval pharyngeal GRNs is involved in mediating both avoidance and suppression of ingestion. Our results show that feeding behavior is coordinated by a multistep regulatory process employing relatively independent neural elements. These findings are consistent with a model in which distinct sensory pathways act as modulatory circuits controlling distinct subprograms during feeding.
    Type of Medium: Online Resource
    ISSN: 2373-2822
    Language: English
    Publisher: Society for Neuroscience
    Publication Date: 2020
    detail.hit.zdb_id: 2800598-3
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  • 5
    Online Resource
    Online Resource
    eLife Sciences Publications, Ltd ; 2019
    In:  eLife Vol. 8 ( 2019-11-20)
    In: eLife, eLife Sciences Publications, Ltd, Vol. 8 ( 2019-11-20)
    Abstract: Cell diversity of the brain and how it is affected by starvation, remains largely unknown. Here, we introduce a single cell transcriptome atlas of the entire Drosophila first instar larval brain. We first assigned cell-type identity based on known marker genes, distinguishing five major groups: neural progenitors, differentiated neurons, glia, undifferentiated neurons and non-neural cells. All major classes were further subdivided into multiple subtypes, revealing biological features of various cell-types. We further assessed transcriptional changes in response to starvation at the single-cell level. While after starvation the composition of the brain remains unaffected, transcriptional profile of several cell clusters changed. Intriguingly, different cell-types show very distinct responses to starvation, suggesting the presence of cell-specific programs for nutrition availability. Establishing a single-cell transcriptome atlas of the larval brain provides a powerful tool to explore cell diversity and assess genetic profiles from developmental, functional and behavioral perspectives.
    Type of Medium: Online Resource
    ISSN: 2050-084X
    Language: English
    Publisher: eLife Sciences Publications, Ltd
    Publication Date: 2019
    detail.hit.zdb_id: 2687154-3
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  • 6
    Online Resource
    Online Resource
    eLife Sciences Publications, Ltd ; 2021
    In:  eLife Vol. 10 ( 2021-12-03)
    In: eLife, eLife Sciences Publications, Ltd, Vol. 10 ( 2021-12-03)
    Abstract: Despite the small number of gustatory sense neurons, Drosophila larvae are able to sense a wide range of chemicals. Although evidence for taste multimodality has been provided in single neurons, an overview of gustatory responses at the periphery is missing and hereby we explore whole-organ calcium imaging of the external taste center. We find that neurons can be activated by different combinations of taste modalities, including opposite hedonic valence and identify distinct temporal dynamics of response. Although sweet sensing has not been fully characterized so far in the external larval gustatory organ, we recorded responses elicited by sugar. Previous findings established that larval sugar sensing relies on the Gr43a pharyngeal receptor, but the question remains if external neurons contribute to this taste. Here, we postulate that external and internal gustation use distinct and complementary mechanisms in sugar sensing and we identify external sucrose sensing neurons.
    Type of Medium: Online Resource
    ISSN: 2050-084X
    Language: English
    Publisher: eLife Sciences Publications, Ltd
    Publication Date: 2021
    detail.hit.zdb_id: 2687154-3
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  • 7
    Online Resource
    Online Resource
    Springer Science and Business Media LLC ; 2020
    In:  Scientific Reports Vol. 10, No. 1 ( 2020-02-17)
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 10, No. 1 ( 2020-02-17)
    Abstract: Organisms possess an endogenous molecular clock which enables them to adapt to environmental rhythms and to synchronize their metabolism and behavior accordingly. Circadian rhythms govern daily oscillations in numerous physiological processes, and the underlying molecular components have been extensively described from fruit flies to mammals. Drosophila larvae have relatively simple nervous system compared to their adult counterparts, yet they both share a homologous molecular clock with mammals, governed by interlocking transcriptional feedback loops with highly conserved constituents. Larvae exhibit a robust light avoidance behavior, presumably enabling them to avoid predators and desiccation, and DNA-damage by exposure to ultraviolet light, hence are crucial for survival. Circadian rhythm has been shown to alter light-dark preference, however it remains unclear how distinct behavioral strategies are modulated by circadian time. To address this question, we investigate the larval visual navigation at different time-points of the day employing a computer-based tracking system, which allows detailed evaluation of distinct navigation strategies. Our results show that due to circadian modulation specific to light information processing, larvae avoid light most efficiently at dawn, and a functioning clock mechanism at both molecular and neuro-signaling level is necessary to conduct this modulation.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
    detail.hit.zdb_id: 2615211-3
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  • 8
    In: Nature Communications, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2018-10-12)
    Abstract: Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, we reveal that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. We characterise IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. We further demonstrate that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. Our work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2018
    detail.hit.zdb_id: 2553671-0
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  • 9
    Online Resource
    Online Resource
    Springer Science and Business Media LLC ; 2016
    In:  Nature Protocols Vol. 11, No. 12 ( 2016-12), p. 2389-2400
    In: Nature Protocols, Springer Science and Business Media LLC, Vol. 11, No. 12 ( 2016-12), p. 2389-2400
    Type of Medium: Online Resource
    ISSN: 1754-2189 , 1750-2799
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
    detail.hit.zdb_id: 2244966-8
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  • 10
    In: eLife, eLife Sciences Publications, Ltd, Vol. 6 ( 2017-08-08)
    Abstract: Visual systems transduce, process and transmit light-dependent environmental cues. Computation of visual features depends on photoreceptor neuron types (PR) present, organization of the eye and wiring of the underlying neural circuit. Here, we describe the circuit architecture of the visual system of Drosophila larvae by mapping the synaptic wiring diagram and neurotransmitters. By contacting different targets, the two larval PR-subtypes create two converging pathways potentially underlying the computation of ambient light intensity and temporal light changes already within this first visual processing center. Locally processed visual information then signals via dedicated projection interneurons to higher brain areas including the lateral horn and mushroom body. The stratified structure of the larval optic neuropil (LON) suggests common organizational principles with the adult fly and vertebrate visual systems. The complete synaptic wiring diagram of the LON paves the way to understanding how circuits with reduced numerical complexity control wide ranges of behaviors.
    Type of Medium: Online Resource
    ISSN: 2050-084X
    Language: English
    Publisher: eLife Sciences Publications, Ltd
    Publication Date: 2017
    detail.hit.zdb_id: 2687154-3
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