Behavioral Assays Research Articles

Therapeutic potential of mackerel-derived peptides and the synthetic tetrapeptide TVGF for sleep disorders in a light-induced anxiety zebrafish model

IntroductionAnxiety-like insomnia is a known risk factor for the onset and worsening of certain neurological diseases, including Alzheimer’s disease. Due to the adverse effects of current anti-insomnia medications, such as drug dependence and limited safety, researchers are actively exploring natural bioactive compounds to mitigate anxiety-like insomnia with fewer side effects. Mackerel (Pneumatophorus japonicus), a traditional Chinese medicine, is known for its tonic effects and is commonly used to treat neurasthenia. The use of mackerel protein extract has been shown to effectively improve symptoms of light-induced anxiety-like insomnia in a zebrafish model.MethodsThis study examines the effects of mackerel bone peptides (MW < 1 kDa, MBP1) and the synthetic peptide Thr-Val-Gly-Phe (TVGF) on light-induced anxiety-like insomnia in zebrafish. The evaluation is conducted through behavioral observation, biochemical marker analysis, and gene transcriptome profiling.ResultsMBP1 significantly alleviated abnormal hyperactivity and restored neurotransmitter levels (dopamine and γ-aminobutyric acid) to normal. Moreover, it mitigated oxidative stress by reducing reactive oxygen species production and malonaldehyde levels, while enhancing antioxidant enzyme activities (superoxide dismutase and catalase). This was further attributed to the regulation of lipid accumulation and protein homeostasis. Furthermore, MBP1 ameliorated sleep disturbances primarily by restoring normal expression levels of genes involved in circadian rhythm (per2 and sik1) and visual function (opn1mw2, zgc:73075, and arr3b). Molecular docking analysis indicated that TVGF exhibited good affinity for receptors linked to sleep disturbances, including IL6, HTR1A, and MAOA. TVGF exhibited sedative effects in behavioral assays, mainly mediated by regulating the normal expression of genes associated with circadian rhythm (cry1bb, cry1ba, per2, per1b and sik1), visual function (opn1mw1, gnb3b, arr3b, gnat2), purine metabolism (pnp5a), and stress recovery (fkbp5).DiscussionThese findings suggest that MBP1 and TVGF could be promising therapies for light-induced anxiety-like insomnia in humans, offering safer alternatives to current medications. Additionally, the regulation of genes related to circadian rhythm and visual perception may be a key mechanism by which MBP1 and TVGF effectively relieve anxiety-like insomnia.

CNSC-13. CROSSTALK BETWEEN SENSORY NEURONAL ACTIVITY AND TUMOR CELLS PROMOTES MALIGNANCY AND CANCER PAIN

Abstract BACKGROUND Nerve infiltration of solid tumors is associated with poor prognosis in multiple cancer types. Understanding the role of neuronal activity in cancer progression offers innovative approaches for treating malignancies and cancer-associated neurological issues such as pain. Pain perception is mediated by the activity of peripheral sensory neurons. Many cancer types induce pain, and sensory innervation could support tumor growth, raising the interesting hypothesis that cancer pain-associated neuronal activity promotes malignancies. Our study aims to test the hypothesis using malignant peripheral nerve sheath tumor (MPNST) as the experimental platform. METHODS in vivo allograft models were established by implanting mouse MPNST cells into the sciatic nerves of male and female mice, followed by performing pain behavior assays (von Frey, Hargreaves, and conditioned place preference tests) and tumor growth measurements. Primary mouse dorsal root ganglion (DRG) sensory neuron cultures were prepared for calcium imaging and neuron-tumor co-culture experiments. RESULTS MPNST tumor-bearing mice exhibited hypersensitivity to noxious and non-noxious stimuli (evoked pain) and ongoing pain, compared to the sham controls. MPNST-conditioned media increased intracellular calcium concentration and ATF3 (indicator for nerve injury/stress) expression in primary sensory neuron culture, suggesting that MPNST tumor cell secretory factors may induce pain by triggering sensory neuron injury/stress responses that increase sensory neuron activity/sensitivity. Reciprocally, we found that stimulation of sensory neuron activity accelerates MPNST tumor growth. The conditioned media from stimulated sensory neurons increased tumor proliferation (EdU assay) in vitro, suggesting that sensory neuron activity-induced paracrine factors increase MPNST cell growth. CONCLUSIONS MPNST is a devastating cancer that grows within peripheral nerves which induce intractable pain. Our findings demonstrate a feedforward cycle where the cancer cells induce sensory neuron hypersensitivity/hyperactivity (pain) which in turn further accelerates tumor growth. Future studies will identify the molecular mechanisms underlying this crosstalk between MPNST and sensory neuron activity.

Guidelines for anti‐obesity assays in Drosophila melanogaster model

AbstractAccumulating evidence showed that obesity leads to severe and various diseases chronically. Although the long list of mechanisms that obesity can harm us is still elongating, more rapid animal models are desired to investigate and illustrate obesity and help us to screen potential treatments. Drosophila melanogaster compared to other commonly used model for in vivo investigations shares many homologous genes and has been used for anti‐obesity research. In this guideline, we summarized the most commonly used assays containing behavioral assays, quantification of body fat, and quantification of glucose, transcriptional activation of genes assays and discussed the future work for anti‐obesity research with D. melanogaster to introduce it as a potential model organism for short‐term anti‐obesity treatment investigations.

Zebrafish as a model to study PERK function in developmental diseases: implications for Wolcott-Rallison syndrome.

Developmental diseases are challenging to investigate due to their clinical heterogeneity and relatively low prevalence. The Wolcott-Rallison Syndrome (WRS) is a rare developmental disease characterized by skeletal dysplasia and permanent neonatal diabetes due to loss-of-function mutations in the endoplasmic reticulum stress kinase PERK (EIF2AK3). The lack of efficient and less invasive therapies for WRS highlights the need for new animal models that replicate the complex pathological phenotypes, while preserving scalability for drug screening. Zebrafish exhibits high fecundity and rapid development that facilitate efficient and scalable in vivo drug testing. Here, we aimed to assess the potential of zebrafish to study PERK function and its pharmacological modulation, and as model organism of developmental diseases such as the WRS. Using bioinformatic analyses, we showed high similarity between human and zebrafish PERK. We used the pharmacological PERK inhibitor GSK2606414, which was bioactive in zebrafish, to modulate PERK function. Using transgenic zebrafish expressing fluorescent pancreatic markers and a fluorescent glucose probe, we observed that PERK inhibition decreased β cell mass and disrupted glucose homeostasis. By combining behavioural and functional assays, we show that PERK-inhibited zebrafish present marked skeletal defects and defective growth, as well as neuromuscular and cardiac deficiencies, which are clinically relevant in WRS patients, while sparing parameters like otolith area and eye/body ratio which are not associated with WRS. These results show that zebrafish holds potential to study PERK function and its pharmacological modulation in developmental disorders like WRS, assisting research on their pathophysiology and experimental treatments.

NompC regulates locomotion and touch sensation in Bactrocera dorsalis.

No mechanoreceptor potential C (NompC) is a major mechanotransduction channel with an important role in sensing of external mechanical stimuli by insects, which help these organisms to avoid injury and adapt to environmental changes. To explore the biological functions of NompC in Bactrocera dorsalis, a notorious agricultural pest, we successfully generated NompC knockout strains using clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) technology. BdorNompC knockout led to an adult lethal phenotype, with approximately 100% mortality at 3 d after eclosion. Morphological observation revealed that the legs and wings of BdorNompC knockout insects were deformed, while behavioral assays showed that the locomotion was impaired in both adults and larvae, relative to that of the wild-type strain. Moreover, BdorNompC knockout reduced gentle-touch response in larvae. These results suggest that BdorNompC is critical for B. dorsalis survival, and that this mechanosensation channel represents a potential new target for pest control agents. Our findings also represent novel evidence indicating that insect NompC is involved in modulating adult wing and leg morphology.

AAVrh.10 Delivery of Novel APOE2-Christchurch Variant Suppresses Amyloid and Tau Pathology in Alzheimer’s Disease Mice

Gene therapy to treat hereditary disorders conventionally delivers the normal allele to compensate for loss-of-function mutations. More effective gene therapy may be achieved using a gain-of-function variant. We tested the hypothesis that AAVrh.10-mediated CNS delivery of the human APOE2 allele with the Christchurch mutation (R136S) (“E2Ch”), will provide superior protection against APOE4-associated Alzheimer’s disease in mice compared to the unmodified APOE2 allele (“E2”). The vectors were assessed in two mouse strains with humanized APOE4: APP.PSEN1/TRE4 “amyloid mice” and P301S/TRE4, “tau mice”. Both the E2Ch and E2 vectors prevented Aβ42 and Aβ40 accumulation and decreased β-amyloid aggregates in amyloid mice. But only the E2Ch vector suppressed decreased tau tangles in tau mice. Microglial activation and reactive astrocytes were significantly suppressed by both vectors in amyloid mice but only the E2Ch vector mediated significant suppression of Iba1 and GFAP in tau mice. In four behavioral assays, the E2 and E2Ch vectors had similar benefits in amyloid mice, but E2Ch outperformed E2 in tau mice. In summary, while E2 is effective in suppressing amyloid pathology, the novel E2 variant E2Ch more effectively treats both the amyloid and tau pathology of murine AD in APOE4 background, supporting the development of AAVrh.10APOE2Ch as a therapy for APOE4-associated AD.

Potential advantage of invasive estuarine worms over native species under exposure to relevant concentrations of graphene oxide: Behavioral and biochemical insights.

Technological development using graphene oxide (GO) has increased in the last years, leading to the release of this contaminant to final sinks, such as estuaries. Due to their potential to flocculate and deposit when interacting with high ionic strength media, GO poses a threat, especially to benthic organisms like polychaetes. In addition to chemical contamination, estuaries also face a severe threat from invasive species, which can cause irreversible damage to ecosystems. The combination of abiotic and biotic stressors may work together on native species, decreasing their resilience. Thus, this study aims to assess the effects of an abiotic stressor, GO nanosheets (0.001, 0.01, 0.1, 1, 10mg GO/Kg dw) on Hediste diversicolor (native species) and Arenicola marina (invasive species) through several behavioral assays and biochemical markers. The impact of invasive species A. marina (biotic factor) on H. diversicolor avoidance behavior was also evaluated. Obtained results demonstrated that H. diversicolor fled from lower GO contamination compartments to higher ones and that exposure to increased GO concentrations negatively impacted its burrowing activity. They were unable to escape from higher contamination compartments, but at the highest concentrations, the bioturbation activity was significantly higher, which may indicate that H. diversicolor tended to dwell deeper in the sediment. A. marina showed an escape behavior from compartments with higher GO concentrations. Additionally, this species' bioturbation activity significantly decreased when exposed to GO. Moreover, avoidance tests demonstrated that the presence of A. marina affected the behavior of H. diversicolor. Regarding oxidative stress, H. diversicolor seems to be more impacted than A. marina, since Lipid peroxidation levels were higher in all GO concentrations and Superoxide dismutase activity significantly increased in the lowest GO levels. Overall, H. diversicolor spatial distribution may be severely constrained under abiotic and biotic stress, while A. marina's higher foraging activity may promote its propagation in the estuary. Behavioral tests, combined with biochemical markers have shown to be relevant tools for the development of more environmental-realistic assessment and monitoring frameworks for estuaries.

Olfactory response to HIPVs and biological aspects of Chrysoperla externa intended for the biological control of Aleurodicus cocois in cashew

ABSTRACT This study evaluates the olfactory response of Chrysoperla externa to the volatiles of the Embrapa 51 genotype of the cashew induced by the herbivory of Aleurodicus cocois, identifies the chemical profile of these compounds and evaluates the development of C. externa fed on A. cocois. The volatiles emitted by the leaves of the cashew genotype, both infested and not infested with A. cocois, were analysed by solid-phase microextraction with GC-MS. In the olfactometry tests, C. externa was attracted to and spent more time in the olfactometer arm containing cashew plant volatiles induced by A. cocois. From the analysis of the volatile profiles, α-phellandrene, α-terpinene, ρ-mentha-2,4(8)-diene, β-caryophyllene and germacrene D were released in large amounts by the Embrapa 51 genotype only after infestation by A. cocois; these compounds may therefore be involved in attracting the predator. Under laboratory conditions, although the development time and survival of some immature stages of C. externa may vary with the type of prey, when fed on A. cocois, the predator is able to complete the egg-adult development cycle. Based on the behavioural and biological assays, C. externa is an effective biocontrol agent for A. cocois in the cashew.

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Neurotoxicity Assessment of Amicarbazone Using Larval Zebrafish

Amicarbazone (AMZ), a triazolinone herbicide widely applied in agriculture, is known to inhibit photosystem II in target plants, disrupting photosynthesis and causing oxidative stress that leads to weed mortality. Despite its widespread use, the developmental and neurotoxic effects of AMZ on aquatic organisms remain underexplored. This study assesses the impact of AMZ exposure on zebrafish (Danio rerio) embryos/larvae, focusing on developmental toxicity and neurotoxicity. Zebrafish were exposed to AMZ at various concentrations to evaluate survival, malformations, heart rate, and behavior. Significant developmental defects, including reduced survival rates, increased malformations, and decreased heart rates, were observed in zebrafish embryos exposed to AMZ, particularly at higher concentrations. Additionally, behavioral assays revealed decreased locomotor activity, particularly at concentrations of 100 and 200 mg/L. Moreover, AMZ exposure disrupted motor axon formation, oligodendrocyte development, and the expression of key genes involved in neurodevelopment. The downregulation of cholinergic, dopaminergic, and serotonergic signaling pathways was also identified, indicating neurotoxicity. These findings highlight AMZ’s potential to induce both developmental and neurotoxic effects in zebrafish and suggest the need for further research on its long-term ecological impacts.

Response of hibiscus mealybug (Hemiptera: Pseudococcidae) to citrus volatiles induced by mechanical injury.

Hibiscus mealybug, Nipaecoccus viridis (Newstead) (Hemiptera: Pseudococcidae), is a recent invasive pest of citrus and many other crops in Florida. Nipaecoccus viridis attacks all above ground parts of citrus trees and heavy infestation can cause leaf drop and premature abortion of developing fruits. We quantified greater captures of N. viridis in cardboard band traps on areas of citrus trees that were intentionally injured by mechanical rasping of epidermal tissues as compared with similar but uninjured citrus branches. Direct field collection of headspace volatiles from mechanically injured or intact citrus branches revealed both qualitative and quantitative differences. Certain volatiles (γ-terpinene, citronellal, citronellyl acetate, β-E-farnesene, α-humulene, and α-E-E-farnesene) were only present in samples from damaged citrus branches. Behavioral assays using a laboratory Y-tube olfactometer revealed attraction of N. viridis to volatiles associated with mechanical damage of citrus including synthetic β-ocimene, γ-terpinene, sabinene, isomers of farnesene, and citronellal when loaded into lures at either of 2 concentrations (0.01 or 0.1 µg/µl). Subsequent field trapping experiments confirmed increased captures of various life stages of N. viridis in cardboard band traps baited with a 10.0 µg/µl concentration of farnesene:ocimene:sabinene blend (in 7:13:17 ratio), as well as those releasing either farnesene or ocimene alone at this same concentration, as compared with the mineral oil (diluent) negative control. Our results indicate that common plant related terpenes released by citrus following mechanical damage may be useful for development of an effective monitoring trap for N. viridis.

Prey nutrient content is associated with the trophic interactions of spiders and their prey selection under field conditions

Consumers are thought to select food resources based on their nutritional content. While laboratory experiments have explored this, the nutritional dynamics of invertebrate predators have been scarcely studied in the field given various methodological constraints. The intersection of these nutritional dynamics with predator traits is also poorly characterised, leading to many gaps in our understanding of how different predators forage and feed in natural systems. Here, we integrate dietary metabarcoding with prey macronutrient (protein, lipid and carbohydrate) content and abundance to assess how nutrients and predator traits (sex, life stage and taxonomy) interactively drive prey preferences in the field, using spider–prey interactions as a model system. Different spider genera, sexes and life stages had nutritionally distinct diets. Our analyses demonstrated disproportionate foraging (selection and avoidance) for prey rich in different macronutrients, with the nature of these relationships differing between spider taxa, life stages and sexes. This may be explained by niche differentiation among spider groups, driven by biases toward prey rich in different nutrients, or nutrient‐specific foraging in which individual spiders vary their nutritional preferences to redress deficits, although further evidence is required to confirm this. This insight into the nutritional dynamics of generalist invertebrate predators extends our understanding beyond lab‐based behavioural assays and provides a novel framework for other complex real‐world systems.

A Cortical Site that Encodes Vocal Expression and Reception.

Socially effective vocal communication requires brain regions that encode expressive and receptive aspects of vocal communication in a social context-dependent manner. Here, we combined a novel behavioral assay with microendoscopy to interrogate neuronal activity in the posterior insula (pIns) in socially interacting mice as they switched rapidly between states of vocal expression and reception. We found that distinct but spatially intermingled subsets of pIns neurons were active during vocal expression and reception. Notably, pIns activity during vocal expression increased prior to vocal onset and was also detected in congenitally deaf mice, pointing to a motor signal. Furthermore, receptive pIns activity depended strongly on social cues, including female odorants. Lastly, tracing experiments reveal that deep layer neurons in the pIns directly bridge the auditory thalamus to a midbrain vocal gating region. Therefore, the pIns is a site that encodes vocal expression and reception in a manner that depends on social context.

Multi-Characteristic Opsin Therapy to Functionalize Retina, Attenuate Retinal Degeneration, and Restore Vision in Mouse Models of Retinitis Pigmentosa.

Retinal degeneration 1 and 10 (rd1 and rd10) mice are useful animal models of retinitis pigmentosa (RP) with rapidly and slowly progressive pathologies, respectively. Our study aims were to determine the effect of adeno-associated viral vector 2 (AAV2)-delivered multi-characteristic opsin (MCO-010; under the control of a metabotropic glutamate receptor-6 promoter enhancer) on the morphological and functional characteristics of vision in both rd1 and rd10 mice. Various retinal measures of MCO-010 transduction and electrophysiological, behavioral, and other routine blood analyses were performed in the rd1 and/or rd10 mice after intravitreal injection of 1µL of MCO-010 or AAV2 vehicle. Functional tests included electroretinogram, visually evoked potential, and behavior assay (optomotor and water maze). Retinal thickness, intraocular pressure, and plasma cytokine levels were also determined. Following intravitreal MCO-010 injection, approximately 80% of bipolar cells were transduced in the retina, and no alterations in retinal thickness were observed at 4months post-injection. However, retinal thickness significantly decreased in control mice. MCO-010 treatment increased head movements and induced faster navigation of mice to the platform in a water-maze test. The MCO-010 gene therapy helped preserve visually evoked electrical response in the retina and visual cortex. No ocular toxicity, immunotoxicity, or phototoxicity was observed in the MCO-010-treated mice, even under chronic intense light conditions. Intravitreal MCO-010 was well tolerated in rd1 and rd10 mice models of RP, and it appeared to attenuate retinal photoreceptor degeneration based on retinal structure and functional outcome measures. As reported here, optogenetic treatment of the inner retina attenuates further retinal degeneration in addition to photosensitizing higher order neurons, and this disease-modifying aspect should be evaluated in optogenetic clinical trials.

Housing and Husbandry Factors Affecting Zebrafish (Danio rerio) Novel Tank Test Responses: A Global Multi-Laboratory Study.

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes and global collaborative studies offer a unique solution. This study is the first global collaboration using the zebrafish (Danio rerio) novel tank test, a behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show females exhibit more anxiety-like behavior than males, underscoring sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (e.g., rotifers), mitigated anxiety-like behaviors. Environmental stressors, like noise and transportation, significantly impacted behavior. We recommend standardizing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds, and minimizing stressors to improve zebrafish behavioral study reliability.

Monoallelic de novo variants in DDX17 cause a neurodevelopmental disorder.

DDX17 is an RNA helicase shown to be involved in critical processes during the early phases of neuronal differentiation. Globally, we compiled a case-series of 11 patients with neurodevelopmental phenotypes harbouring de novo monoallelic variants in DDX17. All 11 patients in our case series had a neurodevelopmental phenotype, whereby intellectual disability, delayed speech and language, and motor delay predominated. We performed in utero cortical electroporation in the brain of developing mice, assessing axon complexity and outgrowth of electroporated neurons, comparing wild-type and Ddx17 knockdown. We then undertook ex vivo cortical electroporation on neuronal progenitors to quantitatively assess axonal development at a single cell resolution. Mosaic ddx17 crispants and heterozygous knockouts in Xenopus tropicalis were generated for assessment of morphology, behavioural assays, and neuronal outgrowth measurements. We further undertook transcriptomic analysis of neuroblastoma SH-SY5Y cells, to identify differentially expressed genes in DDX17-KD cells compared to controls. Knockdown of Ddx17 in electroporated mouse neurons in vivo showed delayed neuronal migration as well as decreased cortical axon complexity. Mouse primary cortical neurons revealed reduced axon outgrowth upon knockdown of Ddx17 in vitro. The axon outgrowth phenotype was replicated in crispant ddx17 tadpoles and in heterozygotes. Heterozygous tadpoles had clear neurodevelopmental defects and showed an impaired neurobehavioral phenotype. Transcriptomic analysis identified a statistically significant number of differentially expressed genes involved in neurodevelopmental processes in DDX17-KD cells compared to control cells. We have identified potential neurodevelopment disease-causing variants in a gene not previously associated with genetic disease, DDX17. We provide evidence for the role of the gene in neurodevelopment in both mammalian and non-mammalian species and in controlling the expression of key neurodevelopment genes.

A potential evolutionary trap for the extended phenotype of a nematomorph parasite

Abstract Human activities introduce new environmental cues to wild organisms, leading to maladaptive behavioural and life-history decisions known as the ‘evolutionary trap’. This trap is thought to be a major conservation concern for free-living organisms. However, it has never been studied in endosymbionts, one of the most successful and diverse life forms on Earth. Here, we examine this trap in the extended phenotype of a parasite that exploits the visual system of hosts to alter host behaviour for its benefit. Arboreal mantids infected by nematomorph parasites are drawn to horizontally polarized light, thereby inducing them to enter the water. In this study, we found that the degree of linear polarisation (DOP) of reflected light served as a reliable environmental cue for identifying perennial waters, where nematomorphs can survive in their aquatic life stage without drying out. Infected mantids exhibit attraction to horizontally polarized light with higher DOP in behavioural assays and jumped into pools reflecting light with higher DOP in field experiments. The asphalt road reflected horizontally polarized light closely resembling the polarisation levels observed in perennial waters, likely leading to a higher prevalence of mantids on asphalt roads compared to those found in natural arboreal habitats. In a field experiment, we observed infected mantids walking on asphalt roads more often than on cement roads. These findings imply that evolutionary traps can endanger endosymbionts beyond their hosts that directly perceive environmental cues.

Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

Developmental Syngap1 haploinsufficiency in medial ganglionic eminence-derived interneurons impairs auditory cortex activity, social behavior and extinction of fear memory.

Mutations in SYNGAP1, a protein enriched at glutamatergic synapses, cause intellectual disability associated with epilepsy, autism spectrum disorder and sensory dysfunctions. Several studies showed that Syngap1 regulates the time course of forebrain glutamatergic synapse maturation; however, the developmental role of Syngap1 in inhibitory GABAergic neurons is less clear. GABAergic neurons can be classified into different subtypes based on their morphology, connectivity and physiological properties. Whether Syngap1 expression specifically in Parvalbumin (PV) and Somatostatin (SST)-expressing interneurons, which are derived from the medial ganglionic eminence, plays a role in the emergence of distinct brain functions remains largely unknown. We used genetic strategies to generate Syngap1 haploinsufficiency in a) prenatal interneurons derived from the medial ganglionic eminence, b) in postnatal PV cells and c) in prenatal SST interneurons. We further performed in vivo recordings and behavioral assays to test whether and how these different genetic manipulations alter brain function and behavior in mice of either sex.Mice with prenatal-onset Syngap1 haploinsufficiency restricted to Nkx2.1-expressing neurons show abnormal cortical oscillations and increased entrainment induced by 40Hz auditory stimulation, but lack of stimulus-specific adaptation. This latter phenotype was reproduced in mice with Syngap1 haploinsufficiency restricted to PV, but not SST, interneurons. Prenatal-onset Syngap1 haploinsufficiency in Nkx2.1-expressing neurons led to impaired social behavior and inability to extinguish fear memories; however, neither postnatal PV- nor prenatal SST-specific mutant mice show these phenotypes. We speculate that Syngap1 haploinsufficiency in prenatal/perinatal PV interneurons may contribute to cortical activity and cognitive alterations associated with Syngap1 mutations.Significance statement Mutations in the human gene cause a form of developmental epileptic encephalopathy associated with intellectual disability, autism and sensory dysfunctions. Several studies have shown that in addition to playing a major role in the synaptic maturation and plasticity of forebrain excitatory neurons, Syngap1 affects GABAergic circuit function as well. Forebrain GABAergic neurons can be divided into different subtypes. Whether Syngap1 expression specifically in distinct interneuron populations and during specific developmental time windows plays a role in the emergence of distinct brain functions remains largely unknown. Here, we report that early, pre or perinatal Syngap1 expression in developing GABAergic neurons derived from the medial ganglionic eminence promotes the development of auditory cortex function, social behavior and ability to extinguish fear memories.

Oral fentanyl consumption and withdrawal impairs fear extinction learning and enhances basolateral amygdala principal neuron excitatory-inhibitory balance in male and female mice

The number of opioid overdose deaths has increased over the past several years, mainly driven by an increase in the availability of highly potent synthetic opioids, like fentanyl, in the un-regulated drug supply. Over the last few years, changes in the drug supply, and in particular the availability of counterfeit pills containing fentanyl, have made oral use of opioids a more common route of administration. Here, we used a drinking in the dark (DiD) paradigm to model oral fentanyl self-administration using increasing fentanyl concentrations in male and female mice over 5 weeks. Fentanyl consumption peaked in both female and male mice at the 30 µg/mL dose, with female mice consuming significantly more fentanyl than male mice. Mice consumed sufficient fentanyl such that withdrawal was precipitated with naloxone, with males having increased withdrawal symptoms as compared to females, despite lower pharmacological exposure. We also performed behavioral assays to measure avoidance behavior and reward-seeking during fentanyl abstinence. Female mice displayed reduced avoidance behaviors in the open field assay, whereas male mice showed increased avoidance in the light/dark box assay. Female mice also exhibited increased reward-seeking in the sucrose preference test. Fentanyl-consuming mice of both sexes showed impaired cued fear extinction learning following fear conditioning and increased excitatory synaptic drive and increased excitability of BLA principal neurons. Our experiments demonstrate that long-term oral fentanyl consumption results in wide-ranging physiological and behavioral disruptions. This model could be useful to further study fentanyl withdrawal syndrome and behaviors and neuroplasticity associated with protracted fentanyl withdrawal.