Behavioral Assays Research Articles

TNF-α/TNFR1 activated astrocytes exacerbate depression-like behavior in CUMS mice

Neuroinflammation is considered to be a significant mechanism contributing to depression. Several studies have reported that A1 astrocytes were highly prevalent in human neuroinflammatory and neurodegenerative diseases. However, the precise mechanism by which A1 astrocytes contribute to depression remains unclear. Clinical studies have suggested a correlation between TNF-α, an activator of A1 astrocytes, and the severity of depression. Based on these findings, we hypothesized that TNF-α might worsen depression by activating A1 astrocytes. Our previous studies indicated that Rhodomyrtone (Rho) has the potential to improve depression-like behavior in mice. However, the exact mechanism for this effect has not been fully elucidated. Importantly, it was reported that Rho alleviated skin inflammation in a mouse model of psoriasis by inhibiting the expression of TNF-α. Based on this finding, we hypothesized that rhodomyrtone may exert antidepressant effects by modulating the TNF-α pathway. However, further research is required to investigate and validate these hypotheses, shedding light on the relationships between neuroinflammation, A1 astrocytes, TNF-α, and depression. By obtaining a deeper understanding of the underlying mechanisms, these findings could lead to the development of novel antidepressant strategies that target the TNF-α pathway in the context of neuroinflammation. In vivo, based on the established chronic unpredictable mild stress (CUMS) mouse depression model, we characterized the mechanism of TNF-α and Rho during depression by using several behavioral assays, adeno-associated virus(AAV) transfection, western blotting, immunofluorescence, and other experimental methods. In vitro, we characterized the effect of Rho on inflammation in TNF-α-treated primary astrocytes. TNFR1 expression was significantly increased in the hippocampus of depression-like mice, with increased astrocytes activation and neuronal apoptosis. These processes were further enhanced with increasing levels of TNF-α in the cerebrospinal fluid of mice. However, this process was attenuated by knockdown of TNFR1 and infliximab (Inf; a TNF-α antagonist). Injection of rhodomyrtone decreased the expressions of TNFR1 and TNF-α, resulting in significant improvements in mouse depression-like behaviors and reduction of astrocyte activation. TNF-α could be involved in the pathophysiological process of depression, through mediating astrocytes activation by binding to TNFR1. By blocking this pathway, Rho may be a novel antidepressant.

Pharmacologic profile of ITI-333: a novel molecule for treatment of substance use disorders.

Medications are urgently needed to treat symptoms of drug withdrawal and mitigate dysphoria and psychiatric comorbidities that drive opioid abuse and relapse. ITI-333 is a novel molecule in development for treatment of substance use disorders, psychiatric comorbidities, and pain. Characterize the preclinical profile of ITI-333 using pharmacological, behavioral, and physiological assays. Cell-based assays were used to measure receptor binding and intrinsic efficacy of ITI-333; animal models were employed to assess effects on opioid reinstatement, precipitated oxycodone withdrawal, and drug abuse liability. In vitro, ITI-333 is a potent 5-HT2A receptor antagonist (Ki = 8nM) and a biased, partial agonist atμ-opioid (MOP) receptors (Ki = 11nM; lacking β-arrestin agonism) with lesser antagonist activity at adrenergic α1A (Ki = 28nM) and dopamine D1 (Ki = 50nM) receptors. In vivo, ITI-333 blocks 5-HT2A receptor-mediated head twitch and MOP receptor-mediated effects on motor hyperactivity in mice. ITI-333 alone is a naloxone-sensitive analgesic (mice) which suppresses somatic signs of naloxone-precipitated oxycodone withdrawal (mice) and heroin cue-induced reinstatement responding without apparent tolerance or physical dependence after chronic dosing (rats). ITI-333 did not acutely impair gastrointestinal or pulmonary function (rats) and was not intravenously self-administered by heroin-maintained rats or rhesus monkeys. ITI-333 acts as a potent 5-HT2A receptor antagonist, as well a biased MOP receptor partial agonist with low intrinsic efficacy. ITI-333 mitigates opioid withdrawal/reinstatement, supporting its potential utility as a treatment for OUD.

Unravelling the microplastic menace: different polymers additively increase bee vulnerability

Microplastics (MPs) are growing and ubiquitous environmental pollutants and represent one of the greatest contemporary challenges caused by human activities. Current research has predominantly examined the singular toxicological effects of individual polymers, neglecting the prevailing reality of organisms confronted with complex contaminant mixtures and potential synergistic effects. To fill this research gap, we investigated the lethal and sublethal effects of two common MPs, polystyrene (PS - 4.8-5.8 μm) and poly(methyl methacrylate) (PMMA - 1-40 μm), and their combination (MIX), on the pollinating insect Apis mellifera. For each treatment, we evaluated the oral toxicity of two ecologically relevant and one higher concentration (0.5, 5 and 50 mg/L) and analysed their effects on the immune system and worker survival. As immune activation can alter the cuticular hydrocarbon profile of honey bees, we used gas chromatography-mass spectrometry (GC-MS) to investigate whether MPs lead to changes in the chemical profile of foragers and behavioural assay to test whether such changes affect behavioural patterns of social recognition, undermining overall colony integrity. The results indicate an additive negative effect of PS and PMMA on bee survival and immune response, even at ecologically relevant concentrations. Furthermore, alterations in cuticle profiles were observed with both MPs at the highest and intermediate concentrations, with PMMA being mainly responsible. Both MPs exposure resulted in a reduction in the abundance of several cuticular compounds. Hive entry guards did not show increased inspection or aggressive behaviour towards exposed foragers, allowing them to enter the colony without being treated differently from uncontaminated foragers. These findings raise concerns not only for the health of individual bees, but also for the entire colony, which could be at risk if contaminated nestmates enter the colony undetected, allowing MPs to spread throughout the hive.

Toxicity Evaluation of Neonicotinoids to Earthworm (Eisenia fetida) Behaviors by a Novel Locomotion Tracking Assay.

Pesticides are substances used for controlling, preventing, and repelling pests in agriculture. Among them, neonicotinoids have become the fastest-growing class of insecticides because of their efficiency in targeting pests. They work by strongly binding to nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, leading to receptor blockage, paralysis, and death. Despite their selectivity for insects, these substances may be hazardous to non-target creatures, including earthworms. Although earthworms may be invasive in some regions like north America, they contribute to the development of soil structure, water management, nutrient cycling, pollution remediation, and cultural services, positively impacting the environment, particularly in the soil ecosystem. Thus, this study aimed to develop a novel earthworm behavior assay since behavior is a sensitive marker for toxicity assay, and demonstrated its application in evaluating the toxicity of various neonicotinoids. Here, we exposed Eisenia fetida to 1 and 10 ppb of eight neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram pestanal, thiacloprid, thiametoxam, and sulfoxaflor) for 3 days to observe their behavior toxicities. Overall, all of the neonicotinoids decreased their locomotion, showed by a reduction of average speed by 24.94-68.63% and increment in freezing time movement ratio by 1.51-4.25 times, and altered their movement orientation and complexity, indicated by the decrement in the fractal dimension value by 24-70%. Moreover, some of the neonicotinoids, which were acetamiprid, dinotefuran, imidacloprid, nitenpyram, and sulfoxaflor, could even alter their exploratory behaviors, which was shown by the increment in the time spent in the center area value by 6.94-12.99 times. Furthermore, based on the PCA and heatmap clustering results, thiametoxam was found as the neonicotinoid that possessed the least pronounced behavior toxicity effects among the tested pesticides since these neonicotinoid-treated groups in both concentrations were grouped in the same major cluster with the control group. Finally, molecular docking was also conducted to examine neonicotinoids' possible binding mechanism to Acetylcholine Binding Protein (AChBP), which is responsible for neurotransmission. The molecular docking result confirmed that each of the neonicotinoids has a relatively high binding energy with AChBP, with the lowest binding energy was possessed by thiametoxam, which consistent with its relatively low behavior toxicities. Thus, these molecular docking results might hint at the possible mechanism behind the observed behavior alterations. To sum up, the present study demonstrated that all of the neonicotinoids altered the earthworm behaviors which might be due to their ability to bind with some specific neurotransmitters and the current findings give insights into the toxicities of neonicotinoids to the environment, especially animals in a soil ecosystem.

Biological Response Following the Systemic Injection of PEG–PAMAM–Rhodamine Conjugates in Zebrafish

Numerous therapeutic and diagnostic approaches used within a clinical setting depend on the administration of compounds via systemic delivery. Biomaterials at the nanometer scale, as dendrimers, act as delivery systems by improving cargo bioavailability, circulation time, and the targeting of specific tissues. Although evaluating the efficacy of pharmacological agents based on nanobiomaterials is crucial, conducting toxicological assessments of biomaterials is essential for advancing clinical translation. Here, a zebrafish larvae model was explored to assess the biocompatibility of poly(amido amine) (PAMAM), one of the most exploited dendrimers for drug delivery. We report the impact of a systemic injection of polyethylene glycol (PEG)-modified G4 PAMAM conjugated with rhodamine (Rho) as a mimetic drug (PEG–PAMAM–Rho) on survival, animal development, inflammation, and neurotoxicity. A concentration- and time-dependent effect was observed on mortality, developmental morphology, and innate immune system activation (macrophages). Significant effects in toxicological indicators were reported in the highest tested concentration (50 mg/mL PEG–PAMAM–Rho) as early as 48 h post-injection. Additionally, a lower concentration of PEG–PAMAM–Rho (5 mg/mL) was found to be safe and subsequently tested for neurotoxicity through behavioral assays. In accordance, no significative signs of toxicity were detected. In conclusion, the dose response of the animal was assessed, and the safe dosage for future use in theragnostics was defined. Additionally, new methodologies were established that can be adapted to further studies in toxicology using other nanosystems for systemic delivery.

Effects of CB1 receptor negative allosteric modulator Org27569 on oxycodone withdrawal symptoms in mice.

Targeting cannabinoid receptor type 1 (CB1R) has shown promise for treating opioid withdrawal symptoms. This study aimed to investigate the efficacy of a specific CB1R negative allosteric modulator (NAM), Org27569, in reducing both naloxone-precipitated and protracted withdrawal symptoms in oxycodone-dependent mice. Mice received escalating doses of oxycodone (9-33mg/kg IP) or saline twice daily for 9 days, followed by a final dose of oxycodone (33mg/kg) or saline in the morning of day 9. In one cohort, the impact of Org27569 (3, 10, and 30mg/kg) on naloxone (10mg/kg IP) precipitated withdrawal symptoms was assessed. In another cohort, Org27569 (3mg/kg) effects on the acquisition of conditioned place aversion to naloxone (0.6mg/kg) precipitated opioid withdrawal, on behaviour following a 7-9-day abstinence period, and on naloxone (0.6mg/kg) precipitated withdrawal-induced escape behaviour in a novel assay were assessed. Although Org27569 decreased opioid withdrawal-induced jumping at doses of 10 and 30mg/kg, these effects were confounded by reduced locomotion. At all doses tested, Org27569 had a modest inhibitory effect on gastrointestinal motility. At the lower dose of 3mg/kg, which was not confounded by locomotor effects, Org27569 did not impact naloxone-precipitated withdrawal-induced jumping, acquisition of oxycodone withdrawal-induced conditioned place aversion, or naloxone-precipitated withdrawal-induced escape behaviour in a novel assay. A clear protracted opioid withdrawal phenotype was not observed in assays of anxiety-like or social behaviour. Org27569 effects on negative affective-like symptoms were confounded by locomotor effects and effects on gastrointestinal motility were not opioid withdrawal specific. Further studies are needed in a model that produces a more pronounced protracted withdrawal syndrome.

Drosophila hydei as a potential vector of Ceratocystis fimbriata, the causal agent of sweetpotato black rot, in storage facilities.

Ceratocystis fimbriata, the causal agent of sweetpotato black rot, is a pathogen capable of developing and spreading within postharvest settings. A survey of North Carolina sweetpotato storage facilities was conducted to determine the arthropods present and identify potential vectors of C. fimbriata. Sixteen taxonomic categories were recovered and the genus Drosophila (Diptera: Drosophilidae) accounted for 79% of individuals sampled with Drosophila hydei (Sturtevant) being the most abundant species. Behavioral assays were conducted to determine if D. hydei is attracted to C. fimbriata inoculated roots and if the pathogen could be recovered from external or internal surfaces of the insect. Flies were released in insect trapping pitchers containing either C. fimbriata inoculated or non-inoculated roots or Petri dishes. No significant differences in fly number were detected in sweetpotato-baited pitchers; however, significant differences were found in the pitcher baited with a mature C. fimbriata culture. Flies were subjected to washes to determine if viable C. fimbriata was present (internally or externally); washes were plated onto carrot agar plates and observed for the presence of C. fimbriata colonies. Both external and internal washes had viable C. fimbriata inoculum with no significant differences, and inoculated sweetpotatoes had a significantly higher number of flies carrying C. fimbriata. This study suggests that D. hydei can carry C. fimbriata from infected sweetpotatoes and move viable C. fimbriata inoculum both externally and internally, making this the first report of any Drosophila spp. serving as a potential vector for the Ceratocystis genus.

Control of polymers’ amorphous-crystalline transition enables miniaturization and multifunctional integration for hydrogel bioelectronics

Soft bioelectronic devices exhibit motion-adaptive properties for neural interfaces to investigate complex neural circuits. Here, we develop a fabrication approach through the control of metamorphic polymers’ amorphous-crystalline transition to miniaturize and integrate multiple components into hydrogel bioelectronics. We attain an about 80% diameter reduction in chemically cross-linked polyvinyl alcohol hydrogel fibers in a fully hydrated state. This strategy allows regulation of hydrogel properties, including refractive index (1.37-1.40 at 480 nm), light transmission (>96%), stretchability (139-169%), bending stiffness (4.6 ± 1.4 N/m), and elastic modulus (2.8-9.3 MPa). To exploit the applications, we apply step-index hydrogel optical probes in the mouse ventral tegmental area, coupled with fiber photometry recordings and social behavioral assays. Additionally, we fabricate carbon nanotubes-PVA hydrogel microelectrodes by incorporating conductive nanomaterials in hydrogel for spontaneous neural activities recording. We enable simultaneous optogenetic stimulation and electrophysiological recordings of light-triggered neural activities in Channelrhodopsin-2 transgenic mice.

Electrophysiological and behavioural responses of fall armyworm Spodoptera frugiperda: A potential phytovolatile technology for its management

The fall armyworm (FAW), scientifically known as Spodoptera frugiperda (J.E. Smith) (Lepidoptera:Noctuidae), is an invasive and polyphagous pest associated with maize (Zea mays L.). An experiment was conducted during 2020–21 at South Eastern University of Sri Lanka, Oluvil, Sri Lanka and the Coconut Research Institute of Sri Lanka Lunuwila, Sri Lanka to investigate the olfactory perception of 22 host plant volatiles by electroantennogram (EAG) and behavioural assays for males, virgin and mated females of FAW. The findings indicated that trans-2-Hexen-1-ol, Benzaldehyde, Nonanoic acid, cis-3-Hexen-1-ol, and 4-Phenyl-2-butanone elicited elevated EAG responses in FAW antennae. Additionally, β-Citronellol and ethyl butyrate elicited amplified responses specifically in mated females and males, respectively. Dose-response study revealed that the observed responses remained statistically non-significant (P>0.05), regardless gender or physiological condition of females across the 1%, 5%, 10% and 15% dose intervals. In behavioural study, observed that 1% Benzaldehyde attracted a substantial number of moths to the treatment side for both virgin and mated females, whereas 1% β-Citronellol displayed repellent properties specifically for mated females of FAW. Therefore, these findings suggest that volatile plant compounds may serve as a theoretical basis for devising novel control strategies for FAW. This includes the potential development of attractants, repellents, and mass capture killers effective for both males and females of FAW, in addition to the currently used female sex pheromone.

Pramipexole improves depression-like behavior in diabetes mellitus with depression rats by inhibiting NLRP3 inflammasome-mediated neuroinflammation and preventing impaired neuroplasticity

BackgroundDiabetes mellitus (DM) is frequently associated with the occurrence and development of depression, and the co-occurrence of diabetes mellitus with depression (DD) may further reduce patients' quality of life. Recent research indicates that dopamine receptors (DRs) play a crucial role in immune and metabolic regulation. Pramipexole (PPX), a D2/3R agonist, has demonstrated promising neuroprotective and immunomodulatory effects. Nevertheless, the therapeutic effects and mechanisms of action of PPX on DM-induced depression are not clear at present. MethodsDepression, DM, and DD were induced in a rat model through a combination of a high-fat diet (HFD) supplemented with streptozotocin (STZ) and chronic unpredictable mild stress (CUMS) combined with solitary cage rearing. The pathogenesis of DD and the neuroprotective effects of DRs agonists were investigated using behavioral assays, enzyme-linked immunosorbent assay (ELISA), hematoxylin-eosin (HE) staining, Nissl staining, Western blotting (WB) and immunofluorescence (IF). ResultsDD rats exhibited more severe dopaminergic, neuroinflammatory, and neuroplastic impairments and more pronounced depressive behaviors than rats with depression alone or DM. Our findings suggest that DRs agonists have significant therapeutic effects on DD rats and that PPX improved neuroplasticity and decreased neuroinflammation in the hippocampus of DD rats while also promoting DG cell growth and differentiation, ultimately mitigating depression-like behaviors. LimitationOur study is based on a rat model. Further evidence is needed to determine whether the therapeutic effects of PPX apply to patients suffering from DD. ConclusionsNeuroinflammation mediated by damage to the dopaminergic system is one of the key pathogenic mechanisms of DD. We provide evidence that PPX has a neuroprotective effect on the hippocampus in DD rats and the mechanism may involve the inhibition of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation by DRs to attenuate the neuroinflammatory response and neuroplasticity damage.

Social buffering of behavioural stress response in two fish species, Nile tilapia (Oreochromis niloticus) and koi carp (Cyprinus carpio)

AbstractThe presence of conspecifics plays a crucial role in mitigating stress responses in social teleost species and holds potential for enhancing welfare in captive fish. While studies on social buffering effects have typically focussed on single species, marked interspecific differences can exist. Here, we conducted an analysis of social buffering of stress response in two of the most extensively farmed fish, the Nile tilapia and the koi carp. Subjects were exposed to a behavioural stress response assay (open‐field test) in three conditions simulating increasing levels of social enrichment: isolation, pairs, or shoals of five fish. We obtained five stress indicators from the assay: thigmotaxis, freezing, activity, erratic movements and interindividual distance in conditions with more than one fish. In both species, erratic movements significantly decreased with increasing levels of social enrichment, suggesting a similar social buffering effect. However, other indicators revealed species differences. Koi carp, but not Nile tilapia, showed a socially‐mediated reduction in thigmotaxis, whereas Nile tilapia, but not Koi carp, showed a socially‐mediated reduction in freezing behaviour. Furthermore, social enrichment determined opposite effects on the activity of the two species: Nile tilapia were more active as group size increased, whereas the opposite trend was found in koi carp. Finally, Nile tilapia showed increased interindividual distance with increasing social group size, whereas no changes were observed for koi carp. Our study indicates that the buffering effects of social enrichment on the behavioural stress response do not completely overlap between different fish species, highlighting the importance of developing finely‐tuned species‐specific enrichments and welfare indicators.

Stimulator of Interferon Genes Pathway Activation through the Controlled Release of STINGel Mediates Analgesia and Anti-Cancer Effects in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) presents significant treatment challenges due to its poor survival and intense pain at the primary cancer site. Cancer pain is debilitating, contributes to diminished quality of life, and causes opioid tolerance. The stimulator of interferon genes (STING) agonism has been investigated as an anti-cancer strategy. We have developed STINGel, an extended-release formulation that prolongs the availability of STING agonists, which has demonstrated an enhanced anti-tumor effect in OSCC compared to STING agonist injection. This study investigates the impact of intra-tumoral STINGel on OSCC-induced pain using two separate OSCC models and nociceptive behavioral assays. Intra-tumoral STINGel significantly reduced mechanical allodynia in the orofacial cancer model and alleviated thermal and mechanical hyperalgesia in the hind paw model. To determine the cellular signaling cascade contributing to the antinociceptive effect, we performed an in-depth analysis of immune cell populations via single-cell RNA-seq. We demonstrated an increase in M1-like macrophages and N1-like neutrophils after STINGel treatment. The identified regulatory pathways controlled immune response activation, myeloid cell differentiation, and cytoplasmic translation. Functional pathway analysis demonstrated the suppression of translation at neuron synapses and the negative regulation of neuron projection development in M2-like macrophages after STINGel treatment. Importantly, STINGel treatment upregulated TGF-β pathway signaling between various cell populations and peripheral nervous system (PNS) macrophages and enhanced TGF-β signaling within the PNS itself. Overall, this study sheds light on the mechanisms underlying STINGel-mediated antinociception and anti-tumorigenic impact.

Volatile chemical cues emitted by an agricultural companion plant (Cnidium monnieri) attract predatory lacewings (Chrysoperla sinica)

Plant volatiles play an important role in the recruitment of arthropod natural enemies and are widely used in foraging by many species. Therefore, identification of plants that release useful volatile organic compounds (VOCs) for influencing natural enemies and for the control of pests can be helpful in conservation biological control. Cnidium monnieri (L.) can convene many predatory enemies (lady beetles, hoverflies, lacewings) as a functional plant. The Chrysoperla sinica (Tjeder) is a dominant generalist predator, and can control the population of various pests in agricultural fields. However, how C. monnier recruits C. sinica and the chemical association between them is unknown. In this study, we tested the attractiveness of odor released by flowers and leaves of C. monnieri to C. sinica in Y-tube olfactometer. Next, we conducted solid phase microextraction (SPME), gas chromatography-mass spectrometry (GC–MS), and electrophysiological studies (EAG) to analyze the compounds and identified the volatile components which have effects on C. sinica. We also used a Y-tube olfactometer to verify the effectiveness of the volatile components on C. sinica under laboratory conditions. In olfactometer trials, C. sinica adults preferred flowers compared with leaves and fresh air. In GC–MS and electroantennography trials, seventy-six volatiles extracted from C. monnieri, twelve antenna-active components in three different concentrations (0.1 μg/mL, 1 μg/mL, 10 μg/mL) were detected for electroantennography. In the last behavioral identification assays, four synthetic compounds (10 μg/mL) had significant attractiveness of adult C. sinica antenna (i.e., γ-terpinene, trans-β-Ocimene, nerolidol, (Z)-3-Hexen-1-ol). These studies suggest that there is a chemical association between natural enemies C. sinica and the functional plant C. monnieri. This study provides a basis that can facilitate the production and application of attracting natural enemy and development and application of enhancing attractants.

Sustained insulin treatment restoring metabolic status, body weight, and cognition in an anorexia nervosa-like animal model in mice

IntroductionAnorexia Nervosa (AN) is a psycho-socio-biological disease characterized by severe weight loss as result of dieting and hyperactivity. Effective treatments are scarce, despite its significant prevalence and mortality. AN patients show lower basal insulin levels and increased metabolic clearance, leading to weight loss, cognitive deficits, and hormonal imbalances. Low-dose polymer insulin could potentially reverse these effects by restoring brain function, reducing fear of weight gain, encouraging food intake, and restoring fat depots. This study evaluates an insulin delivery system designed for sustained release and AN treatment. MethodsAN-like model was established through dietary restriction (DR). On days 1–25, mice were on DR, and on days 26–31 they were on ad libitum regimen. An insulin-loaded delivery system was administered subcutaneously (1% w/w insulin). The impact of insulin treatment on gene expression in the hippocampus (cognition, regulation of stress, neurogenesis) and hypothalamus (eating behavior, mood) was assessed. Behavioral assays were conducted to evaluate motor activity and cognitive function. ResultsThe delivery system demonstrated sustained insulin release, maintaining therapeutic plasma levels. Diet restriction mice treated with the insulin delivery system showed body weight restoration. Gene expression analysis revealed enhanced expression of CB1 and CB2 genes associated with improved eating behavior and cognition, while POMC expression was reduced. Insulin-polymer treatment restored cognitive function and decreased hyperactivity in the AN-like model. ConclusionThe PSA-RA-based insulin delivery system effectively restores metabolic balance, body weight, and cognitive function in the AN model. Its ability to steadily release insulin makes it a promising candidate for AN treatment."

Low-Efficacy Mu Opioid Agonists as Candidate Analgesics: Effects of Novel C-9 Substituted Phenylmorphans on Pain-Depressed Behavior in Mice.

Low efficacy mu opioid receptor (MOR) agonists may serve as novel candidate analgesics with improved safety relative to high-efficacy opioids. This study used a recently validated assay of pain-depressed behavior in mice to evaluate a novel series of MOR-selective C9-substituted phenylmorphan opioids with graded MOR efficacies. Intraperitoneal injection of dilute lactic acid (IP acid) served as a noxious stimulus to depress locomotor activity by mice in an activity chamber composed of two compartments connected by an obstructed door. Behavioral measures included (1) crosses between compartments (vertical activity over the obstruction) and (2) movement counts quantified as photobeam breaks summed across compartments (horizontal activity). Each drug was tested alone and as a pretreatment to IP acid. A charcoal-meal test and whole-body-plethysmography assessment of breathing in 5% CO2 were also used to assess gastrointestinal (GI) inhibition and respiratory depression, respectively. IP acid produced a concentration-dependent depression in crosses and movement that was optimally alleviated by intermediate- to low-efficacy phenylmorphans with sufficient efficacy to produce analgesia with minimal locomotor disruption. Follow-up studies with two low-efficacy phenylmorphans (JL-2-39 and DC-1-76.1) indicated that both drugs produced naltrexone-reversible antinociception with a rapid onset and a duration of ~1hr. Potency of both drugs increased when behavior was depressed by a lower IP-acid concentration, and neither drug alleviated behavioral depression by a non-pain stimulus (IP lithium chloride). Both drugs produced weaker GI inhibition and respiratory depression than fentanyl and attenuated fentanyl-induced GI inhibition and respiratory depression. Results support further consideration of selective, low-efficacy MOR agonists as candidate analgesics. Significance Statement This study used a novel set of mu opioid receptor (MOR)-selective opioids with graded MOR efficacies to examine the lower boundary of MOR efficacy sufficient to relieve pain-related behavioral depression in mice. Two novel low-efficacy opioids (JL-2-39, DC-1-76.1) produced effective antinociception with improved safety relative to higher- or lower-efficacy opioids, and results support further consideration of these and other low-efficacy opioids as candidate analgesics.

Sexual conflict and social networks in bed bugs: effects of social experience.

Living in groups can provide essential experience that improves sexual performance and reproductive success. While the effects of social experience have drawn considerable scientific interest, commonly used behavioral assays often do not capture the dynamic nature of interactions within a social group. Here, we conducted 3 experiments using a social network framework to test whether social experience during early adulthood improves the sexual competence of bed bugs (Cimex lectularius) when placed in a complex and competitive group environment. In each experiment, we observed replicate groups of bed bugs comprising previously socialized and previously isolated individuals of the same sex, along with an equal number of standardized individuals of the opposite sex. Regardless of whether we controlled for their insemination history, previously isolated males mounted and inseminated females at significantly higher rates than previously socialized males. However, we found no evidence of social experience influencing our other measures of sexual competence: proportion of mounts directed at females, ability to overcome female resistance, and strength of opposite-sex social associations. We similarly did not detect effects of social experience on our female sexual competence metrics: propensity to avoid mounts, rate of successfully avoiding mounts, opposite-sex social association strength, and rate of receiving inseminations. Our findings indicate that early social experience does not improve sexual competence in male and female bed bugs.

STEERING THE MICROBIOTA-GUT-BRAIN AXIS BY ANTIBIOTICS TO MODEL NEURO-IMMUNE-ENDOCRINE DISORDERS.

Over the last century, animal models have been employed to study the gut-brain axis and its relationship with physiological processes, including those necessary for survival, such as food intake regulation and thermoregulation; those involved in diseases, ranging from inflammation to obesity; and those concerned to the development of neurodegenerative diseases and neuropsychiatric disorders, such as Alzheimer's disease and autism spectrum disorder, respectively. The gut microbiota has been recognized in the last decade as an essential functional component of this axis. Many reports demonstrate that the gut microbiota influences the development of a vast array of physiological processes. Experiments that use animal models to assess the effect of the gut microbiota on the brain and behavior may involve the acute or chronic administration of wide-spectrum antibiotics. This narrative review summarizes the beneficial or detrimental effects of antibiotics administered prenatally or postnatally to rodents during acute or chronic periods in a wide range of protocols. These include animal models of disease and behavioral paradigms of learning and memory, anxiety, obsessive-compulsive disorder, and autism spectrum disorder. Biomarkers and behavioral assays associated with antibiotic exposure are also included in this review.

Total ginsenosides extend healthspan of aging Drosophila by suppressing imbalances in intestinal stem cells and microbiota

BackgroundDisruption of stem cell and microbial homeostasis accelerates the aging process. Hence, maintaining these balances effectively delays aging and alleviates the symptoms of age-related diseases. Recent research indicates that targeting endoplasmic reticulum (ER) stress and immune deficiency (IMD) signalling may play a positive role in maintaining homeostasis in aging intestinal stem cells (ISC) and microbial equilibrium. Previous research has suggested that total ginsenosides (TG) derived from Panax ginseng C. A. Meyer may exhibit potential anti-aging properties by mitigating ER stress and mediating the IMD pathway. Nevertheless, it remains unclear whether TG improve ISC and microbial homeostasis by modulating ER stress and the IMD pathway to promote healthy aging. PurposeTo elucidate whether TG promotes healthspan in Drosophila and its underlying molecular mechanisms, focusing on its role in regulating ER stress and the IMD pathway to maintain ISC and intestinal microbiota homeostasis. MethodsHigh performance liquid chromatography was performed to detect the main saponin monomer in TG. Survival rate, gut length, barrier function, and feeding/excretion behaviour assays were used to evaluate the effects of TG on the lifespan and gut health of Drosophila. At the stem cell level, “esg-luciferase” reporter system, esg-GFP/delta stem cell fluorescent labelling, and phospho-histone H3+ mitotic activity assays were employed to determine whether TG prevented natural aging or oxidative stress-associated ISC over-proliferation in Drosophila. Immunofluorescence staining was used to detect the effects of TG on ER stress during aging. Overexpression or interference of ER stress target genes and their related c-Jun N-terminal kinase (JNK) gene was manipulated using gene editing technology to verify the molecular mechanism by which TG maintains age-related ISC proliferation homeostasis. Molecular docking and isothermal titration calorimetry were used to verify the direct interactions between TG and ER stress target genes. In addition, at the intestinal flora level, 16S rDNA sequencing was used to analyse the effect of TG on the diversity and abundance of Drosophila intestinal flora and the possible functional pathways involved. RT-qPCR was performed to determine whether TG mediated the expression of target genes in the IMD pathway. A dominant bacterial species-specific mono-association analysis were performed to verify whether the effects of TG on IMD target genes and ISC proliferation depended on the direct control of the dominant bacterial species. ResultsOur results suggest that administration of TG delays the decline in gut morphology and function in aging Drosophila. TG prevents age-associated ISC hyperproliferation by inhibiting ER stress IRE1-mediated JNK signaling. Furthermore, oral TG prevented aging-associated ISC and gut microbiota dysbiosis by remodelling the gut microbiota and inhibiting Acetobacter-mediated activation of IMD target genes. ConclusionTG promotes healthy aging by inhibiting the excessive proliferation of ISC and alleviating intestinal microbial imbalance, thereby providing new insights for the research and development of anti-aging TG products.

Hymenopteran-specific TRPA channel from the Texas leaf cutter ant (Atta texana) is heat and cold activated and expression correlates with environmental temperature.

Leaf cutting ants of the genus Atta cultivate fungal gardens, carefully modifying environmental conditions to maintain optimal temperature for fungal growth. Antennal nerves from Atta are highly temperature sensitive, but the underlying molecular sensor is unknown. Here, we utilize Atta texana (Texas leaf cutter ant) to investigate the molecular basis of ant temperature sensation and how it might have evolved as the range expanded northeast across Texas from ancestral populations in Mexico. We focus on transient receptor potential (TRP) channel genes, the best characterized temperature sensor proteins in animals. Atta texana antennae express 6 of 13 Hymenopteran TRP channel genes and sequences are under a mix of relaxed and intensified selection. In a behavioral assay, we find A. texana workers prefer 24 °C (range 21-26 °C) for fungal growth. There was no evidence of regulatory evolution across a temperature transect in Texas, but instead Hymenoptera-specific TRPA (HsTRPA) expression highly correlated with ambient temperature. When expressed in vitro, HsTRPA from A. texana is temperature activated with Q10 values exceeding 100 on initial exposure to temperatures above 33 °C. Surprisingly, HsTRPA also appears to be activated by cooling, and therefore to our knowledge, the first non-TRPA1 ortholog to be described with dual heat/cold activation and the first in any invertebrate.

Increased Sensitivity in Detection of Deficits Following Two Commonly Used Animal Models of Stroke

Stroke is a leading cause of death and disability in the United States. Most strokes are ischemic, resulting in both cognitive and motor impairments. Animal models of ischemic stroke such as the distal medial cerebral artery occlusion (dMCAO) and photothrombotic stroke (PTS) procedures have become invaluable tools, with their own advantages and disadvantages. The dMCAO model is clinically relevant as it occludes the artery most affected in humans, but yields variability in the infarct location as well as the behavioral and cognitive phenotypes disrupted. The PTS model has the advantage of allowing for targeted location of infarct, but is less clinically relevant. The present study evaluates phenotype disruption over time in mice subjected to either dMCAO, PTS, or a sham surgery. Post-surgery, animals were tested over 28 days on standard motor tasks (grid walk, cylinder, tapered beam, and rotating beam), as well as a novel odor-based operant task; the 5:1 Odor Discrimination Task (ODT). Results demonstrate a significantly greater disturbance of motor control with PTS as compared with Sham and dMCAO. Disruption of the PTS group was detected up to 28 days post-stroke on the grid walk, and up to 7 days on the rotating and tapered beam tasks. PTS also led to significant short-term disruption of ODT performance (1-day post-surgery), exclusively in males, which appeared to be driven by motoric disruption of the lick response. Together, this data provides critical insights into the selection and optimization of animal models for ischemic stroke research. Notably, the PTS procedure was best suited for producing disruptions of motor behavior that can be detected with common behavioral assays and are relatively enduring, as is observed in human stroke.