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Hereditary spastic paraplegias (HSPs) are a diverse set of neurological disorders characterized by progressive spasticity and weakness in the lower limbs caused by damage to the axons of the corticospinal tract. More than 88 genetic mutations have been associated with HSP, yet the mechanisms underlying these disorders are not well understood. We replicated the pathophysiology of one form of HSP known as spastic paraplegia 15 (SPG15) in zebrafish. This disorder is caused in humans by mutations in the ZFYVE26 gene, which codes for a protein called SPASTIZIN. We show that, in zebrafish, the significant reduction of Spastizin caused degeneration of large motor neurons. Motor neuron degeneration is associated with axon demyelination in the spinal cord and impaired locomotion in the spastizin mutants. Our findings reveal that the reduction in Spastizin compromises axonal integrity and affects the myelin sheath, ultimately recapitulating the pathophysiology of HSPs.

In the dynamic landscape of biomedical science, the pursuit of effective treatments for motor neuron disorders like hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA) remains a key priority. Central to this endeavor is the development of robust animal models, with the zebrafish emerging as a prime candidate. Exhibiting embryonic transparency, a swift life cycle, and significant genetic and neuroanatomical congruencies with humans, zebrafish offer substantial potential for research. Despite the difference in locomotion-zebrafish undulate while humans use limbs, the zebrafish presents relevant phenotypic parallels to human motor control disorders, providing valuable insights into neurodegenerative diseases. This review explores the zebrafish's inherent traits and how they facilitate profound insights into the complex behavioral and cellular phenotypes associated with these disorders. Furthermore, we examine recent advancements in high-throughput drug screening using the zebrafish model, a promising avenue for identifying therapeutically potent compounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Garg and Geurten.)

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism., (© 2022. The Author(s).)

Background: The selective aphicide flonicamid is known to cause symptoms in aphids that are like those of chordotonal organ TRPV channel modulator insecticides such as pymetrozine, pyrifluquinazon and afidopyropen. Flonicamid is classified by the Insecticide Resistance Action Committee as a chordotonal organ modulator with an undefined target site. However, although it has been shown not to act on TRPV channels, flonicamid's action on chordotonal organs has not been documented in the literature., Results: Flonicamid causes locusts to extend their hindlegs, indicating an action on the femoral chordotonal organ. In fruit flies, it abolishes negative gravitaxis behavior by disrupting transduction and mechanical amplification in antennal chordotonal neurons. Although flonicamid itself only weakly affects locust chordotonal organs, its major animal metabolite 4-trifluoromethylnicotinamide (TFNA-AM) potently stimulates both locust and fly chordotonal organs. Like pymetrozine, TFNA-AM rapidly increases Ca 2+ in antennal chordotonal neurons in wild-type flies, but not iav 1 mutants, yet the effect is nonadditive with the TRPV channel agonist., Conclusions: Flonicamid is a pro-insecticide form of TFNA-AM, a potent chordotonal organ modulator. The functional effects of TFNA-AM on chordotonal organs of locusts and flies are indistinguishable from those of the TRPV agonists pymetrozine, pyrifluquinazon and afidopyropen. Because our previous results indicate that TFNA-AM does not act directly on TRPV channels, we conclude that it acts upstream in a pathway that leads to TRPV channel activation. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

The analysis of kinematics, locomotion, and spatial tasks relies on the accurate detection of animal positions and pose. Pose and position can be assessed with video analysis programs, the "trackers." Most available trackers represent animals as single points in space (no pose information available) or use markers to build a skeletal representation of pose. Markers are either physical objects attached to the body (white balls, stickers, or paint) or they are defined in silico using recognizable body structures (e.g., joints, limbs, color patterns). Physical markers often cannot be used if the animals are small, lack prominent body structures on which the markers can be placed, or live in environments such as aquatic ones that might detach the marker. Here, we introduce a marker-free pose-estimator (LACE L imbless A nimal tra C k E r) that builds the pose of the animal de novo from its contour. LACE detects the contour of the animal and derives the body mid-line, building a pseudo-skeleton by defining vertices and edges. By applying LACE to analyse the pose of larval Drosophila melanogaster and adult zebrafish, we illustrate that LACE allows to quantify, for example, genetic alterations of peristaltic movements and gender-specific locomotion patterns that are associated with different body shapes. As illustrated by these examples, LACE provides a versatile method for assessing position, pose and movement patterns, even in animals without limbs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Garg, André, Giraldo, Heyer, Göpfert, Dosch and Geurten.)

This protocol enables the quantification of odor-evoked calcium activity in mushroom body Kenyon cells of the Drosophila melanogaster brain at the single bouton level. We also present subsequent characterization of naive and learned odor representations in the context of olfactory coding. This approach to analyzing the neuronal basis of associative learning provides a substrate for similar studies, perhaps in other animals, to probe the attributes of a neuronal memory trace at the level of synapses distributed across neurons. For complete details on the use and execution of this protocol, please refer to Bilz et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

During associative conditioning, animals learn which sensory cues are predictive for positive or negative conditions. Because sensory cues are encoded by distributed neurons, one has to monitor plasticity across many synapses to capture how learned information is encoded. We analyzed synaptic boutons of Kenyon cells of the Drosophila mushroom body γ lobe, a brain structure that mediates olfactory learning. A fluorescent Ca 2+ sensor was expressed in single Kenyon cells so that axonal boutons could be assigned to distinct cells and Ca 2+ could be measured across many animals. Learning induced directed synaptic plasticity in specific compartments along the axons. Moreover, we show that odor-evoked Ca 2+ dynamics across boutons decorrelate as a result of associative learning. Information theory indicates that learning renders the stimulus representation more distinct compared with naive stimuli. These data reveal that synaptic boutons rather than cells act as individually modifiable units, and coherence among them is a memory-encoding parameter., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria . Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria , we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species., (Copyright © 2019 Hahn, Büschgens, Schwedhelm-Domeyer, Bank, Geurten, Neugebauer, Massih, Göpfert and Heinrich.)

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sensing environmental temperatures is essential for the survival of ectothermic organisms. In Drosophila, two of the most used methodologies to study temperature preferences (T P ) and the genes involved in thermosensation are two-choice assays and temperature gradients. Whereas two-choice assays reveal a relative T P , temperature gradients can identify the absolute T p . One drawback of gradients is that small ectothermic animals are susceptible to cold-trapping: a physiological inability to move at the cold area of the gradient. Often cold-trapping cannot be avoided, biasing the resulting T P to lower temperatures. Two mathematical models were previously developed to correct for cold-trapping. These models, however, focus on group behaviour which can lead to overestimation of cold-trapping due to group aggregation. Here we present a mathematical model that simulates the behaviour of individual Drosophila in temperature gradients. The model takes the spatial dimension and temperature difference of the gradient into account, as well as the rearing temperature of the flies. Furthermore, it allows the quantification of cold-trapping and reveals unbiased T P. Additionally, our model reveals that flies have a range of tolerable temperatures, and this measure is more informative about the behaviour than commonly used T P . Online simulation is hosted at http://igloo.uni-goettingen.de . The code can be accessed at https://github.com/zerotonin/igloo .

Animals rely on mechanosensory feedback from proprioceptors to control locomotory body movements. Unexpectedly, we found that this movement control requires visual opsins. Disrupting the Drosophila opsins NINAE or Rh6 impaired larval locomotion and body contractions, independently of light and vision. Opsins were detected in chordotonal proprioceptors along the larval body, localizing to their ciliated dendrites. Loss of opsins impaired mechanically evoked proprioceptor spiking and cilium ultrastructure. Without NINAE or Rh6, NOMPC mechanotransduction channels leaked from proprioceptor cilia and ciliary Inactive (Iav) channels partly disappeared. Locomotion is shown to require opsins in proprioceptors, and the receptors are found to express the opsin gene Rh7, in addition to ninaE and Rh6. Besides implicating opsins in movement control, this documents roles of non-ciliary, rhabdomeric opsins in cilium organization, providing a model for a key transition in opsin evolution and suggesting that structural roles of rhabdomeric opsins preceded their use for light detection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster , an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, we, here, analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.

Moving animals can estimate the distance of visual objects from image shift on their retina (optic flow) created during translational, but not rotational movements. To facilitate this distance estimation, many terrestrial and flying animals perform saccadic movements, thereby temporally separating translational and rotational movements, keeping rotation times short. In this study, we analysed whether a semiaquatic mammal, the harbour seal, also adopts a saccadic movement strategy. We recorded the seals' normal swimming pattern with video cameras and analysed head and body movements. The swimming seals indeed minimized rotation times by saccadic head and body turns, with top rotation speeds exceeding 350 deg s -1 which leads to an increase of translational movements. Saccades occurred during both types of locomotion of the seals' intermittent swimming mode: active propulsion and gliding. In conclusion, harbour seals share the saccadic movement strategy of terrestrial animals. Whether this movement strategy is adopted to facilitate distance estimation from optic flow or serves a different function will be a topic of future research., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)