Light-dark Transition Test Research Articles

Effects of Repeated Administration of Rauwolfia Serpentina on Stress-Induced Anorexia and Leptin Levels: Relationship With Adaptation to Stress

Background: Stress is a major contributor to various health issues, including depression, anorexia nervosa, diabetes, and obesity. Rauwolfia serpentina, a traditional medicinal plant, has shown potential in alleviating stress-induced effects due to its influence on serotonin neurotransmission and its anxiolytic properties. Objective: This study aimed to investigate the effects of repeated administration of Rauwolfia serpentina on stress-induced anorexia and leptin levels, and its relationship with adaptation to stress in rats. Methods: Male albino Wistar rats were divided into four groups: DMSO unstressed, DMSO stressed, Rauwolfia serpentina unstressed, and Rauwolfia serpentina stressed. The stressed groups were subjected to two hours of immobilization daily for five days, followed by oral administration of Rauwolfia serpentina at 10 mg/kg. Behavioral assessments included the open field and light-dark transition tests. Body weight, food intake, and plasma levels of glucose, leptin, and corticosterone were measured. Data were analyzed using ANOVA with post-hoc Newman-Keuls test, and significance was set at p<0.05. Results: Rauwolfia serpentina significantly improved body weight maintenance (98.9 ± 1.3% vs. 99.5 ± 1.7%) and increased food intake (16.3 ± 0.6 g vs. 16.5 ± 0.5 g) in stressed rats. It also reduced anxiety-like behaviors and lowered corticosterone levels (15.9 ± 1.6 ng/mL vs. 18.4 ± 1.7 ng/mL) in stressed groups (p<0.05). Conclusion: Rauwolfia serpentina effectively alleviates stress-induced anorexigenic and anxiogenic effects, suggesting its potential as a therapeutic agent for managing stress-related disorders.

Icariin mitigates anxiety-like behaviors induced by hemorrhagic shock and resuscitation via inhibiting of astrocytic activation

BackgroundAbnormal activation of astrocytes in the amygdala contributes to anxiety after hemorrhagic shock and resuscitation (HSR). Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-associated epigenetic reprogramming of astrocytic activation is crucial to anxiety. A bioactive monomer derived from Epimedium icariin (ICA) has been reported to modulate NF-κB signaling and astrocytic activation. PurposeThe present study aimed to investigate the effects of ICA on post-HSR anxiety disorders and its potential mechanism of action. MethodsWe first induced HSR in mice through a bleeding and re-transfusion model and selectively inhibited and activated astrocytes in the amygdala using chemogenetics. Then, ICA (40 mg/kg) was administered by oral gavage once daily for 21 days. Behavioral, electrophysiological, and pathological changes were assessed after HSR using the light-dark transition test, elevated plus maze, recording of local field potential (LFP), and immunofluorescence assays. ResultsExposure to HSR reduced the duration of the light chamber and attenuated open-arm entries. Moreover, HSR exposure increased the theta oscillation power in the amygdala and upregulated NF-κB p65, H3K27ac, and H3K4me3 expression. Contrarily, chemogenetic inhibition of astrocytes significantly reversed these changes. Chemogenetic inhibition in astrocytes was simulated by ICA, but chemogenetic activation of astrocytes blocked the neuroprotective effects of ICA. ConclusionICA mitigated anxiety-like behaviors induced by HSR in mice via inhibiting astrocytic activation, which is possibly associated with NF-κB-induced epigenetic reprogramming.

Intracranial self-stimulation reverses impaired spatial learning and regulates serum microRNA levels in a streptozotocin-induced rat model of Alzheimer disease.

The assessment of deep brain stimulation (DBS) as a therapeutic alternative for treating Alzheimer disease (AD) is ongoing. We aimed to determine the effects of intracranial self-stimulation at the medial forebrain bundle (MFB-ICSS) on spatial memory, neurodegeneration, and serum expression of microRNAs (miRNAs) in a rat model of sporadic AD created by injection of streptozotocin. We hypothesized that MFB-ICSS would reverse the behavioural effects of streptozotocin and modulate hippocampal neuronal density and serum levels of the miRNAs. We performed Morris water maze and light-dark transition tests. Levels of various proteins, specifically amyloid-β precurser protein (APP), phosphorylated tau protein (pTAU), and sirtuin 1 (SIRT1), and neurodegeneration were analyzed by Western blot and Nissl staining, respectively. Serum miRNA expression was measured by reverse transcription polymerase chain reaction. Male rats that received streptozotocin had increased hippocampal levels of pTAU S202/T205, APP, and SIRT1 proteins; increased neurodegeneration in the CA1, dentate gyrus (DG), and dorsal tenia tecta; and worse performance in the Morris water maze task. No differences were observed in miRNAs, except for miR-181c and miR-let-7b. After MFB-ICSS, neuronal density in the CA1 and DG regions and levels of miR-181c in streptozotocin-treated and control rats were similar. Rats that received streptozotocin and underwent MFB-ICSS also showed lower levels of miR-let-7b and better spatial learning than rats that received streptozotocin without MFB-ICSS. The reversal by MFB-ICSS of deficits induced by streptozotocin was fairly modest. Spatial memory performance, hippocampal neurodegeneration, and serum levels of miR-let-7b and miR-181c were affected by MFB-ICSS under AD-like conditions. Our results validate the MFB as a potential target for DBS and lend support to the use of specific miRNAs as promising biomarkers of the effectiveness of DBS in combatting AD-associated cognitive deficits.

Curcumin analogue NL04 inhibits spinal cord central sensitization in rats with bone cancer pain by inhibiting NLRP3 inflammasome activation and reducing IL-1β production

The management and therapy of bone cancer pain (BCP) remain formidable clinical challenges. Curcumin and its analogues have been shown to have anti-inflammatory and analgesic properties. In the present study, we investigated the efficacy of curcumin analogue NL04 (NL04) in modulating inflammation in spinal dorsal horn (SDH), thereby exploring its potential to reduce central sensitization of BCP in a rat model. Differing doses of NL04 and curcumin were administered intrathecally either once (on day 12 of BCP) or over seven consecutive days (from day 6–12 of BCP). Results indicated that the ED50 for NL04 and curcumin ameliorating BCP-induced mechanical hyperalgesia is 49.08 μg/kg and 489.6 μg/kg, respectively. The analgesic effects at various doses of NL04 lasted between 4 and 8 h, with sustained administration over a week maintaining pain relief for 1–4 days, while also ameliorating locomotor gait via gait analysis and reducing depressive and anxiety-like behaviors via open-field and light–dark transition tests. The analgesic effects at various doses of curcumin lasted 4 h, with sustained administration over a week maintaining pain relief for 0–2 days. ELISA, Western blotting, qPCR, and immunofluorescence assays substantiated that intrathecal administration of NL04 on days 6–12 of BCP dose-dependently lowered spinal IL-1β and IL-18 levels and significantly reduced the expression of IKKβ genes and proteins, as well as the downstream cleavage of the trans-Golgi network (TGN). Whole-cell patch-clamp results demonstrated that NL04 inhibits potassium ion efflux in rat primary spinal neurons. Thus, NL04 exhibits significant analgesic effects in a BCP rat model by downregulating IKKβ expression and inhibiting neuronal potassium ion efflux, which, in turn, suppresses the activation of NLRP3 inflammasomes and reduces IL-1β production, potentially ameliorating pain management in BCP.

Line- and sex-dependent effects of juvenile stress on contextual fear- and anxiety-related behavior in high- and low-alcohol-preferring mouse lines

Juvenile stress (JS) is a known risk factor for the development of alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD), both of which are frequently co-morbid. Data suggest there may be common, genetically-influenced biological responses to stress that contribute to the development of both AUD and PTSD. The present study investigated the impact of JS on contextual fear learning and extinction, as well as corticosterone (CORT) responses before and after JS, before and after contextual fear conditioning (CFC), and after fear extinction in male and female high-alcohol-preferring (HAP2) and low-alcohol-preferring (LAP2) mouse lines. We also measured unconditioned anxiety-related behavior in the light-dark-transition test before CFC. HAP2 and LAP2 mice did not differ in fear acquisition, but HAP2 mice showed faster fear extinction compared to LAP2 mice. No effects of JS were seen in HAP2 mice, whereas in LAP2 mice, JS reduced fear acquisition in males and facilitated fear extinction in females. Females showed greater fear-related behavior relative to males, regardless of subgroup. HAP2 males demonstrated more anxiolytic-like responses than LAP2 males and LAP2 females demonstrated more anxiolytic-like responses than LAP2 males in the light-dark transition test. HAP2 and LAP2 mice did not differ in CORT during the juvenile stage; however, adult LAP2 mice showed greater CORT levels than HAP2 mice at baseline and after CFC and extinction testing. These findings build upon prior work in these unique mouse lines that differ in genetic propensity toward alcohol preference and provide new information regarding contextual fear learning and extinction mechanisms theorized to contribute to co-morbid AUD and PTSD.

Oxytocin receptors in the prefrontal cortex play important roles in short-term social recognition in mice

We examined the roles of oxytocin (OT) receptors in the prefrontal cortex (PFC) in short- and long-term social recognition and anxiety-related behaviors in mice. Mice injected with high or low doses of an OT receptor antagonist (OTA) or vehicle performed the social recognition test, the open-field test, and the light–dark transition test. In the social recognition test, with three daily trials over three consecutive days, control mice showed short-term recognition of a conspecific on all three days. In contrast, a high-dose injection of OTA impaired short-term social recognition on the second and third days, and it was impaired by a low-dose injection of OTA on the third day. These results suggested that OTA injection into the PFC dose-dependently inhibited short-term social recognition within each day. All three groups did not show any long-term social recognition across three days. OTA injection did not affect anxiety related behavior in the open-field and light–dark transition tests. Our findings demonstrated that OT receptors in the PFC played important roles in short-term social recognition.

Sceletium tortuosum-derived mesembrine significantly contributes to the anxiolytic effect of Zembrin®, but its anti-depressant effect may require synergy of multiple plant constituents

Ethnopharmacology relevanceSceletium tortuosum (L.) N.E.Br. (ST) is an alkaloid-rich succulent plant with various mechanisms of action that infer psychotropic effects. These actions correlate with clinical evidence suggesting efficacy in the treatment of depression and anxiety, in line with its use by indigenous populations. Its low side effect profile suggests potential of ST to improve the overall wellbeing and compliance of millions of patients that experience severe side effects and/or do not respond to current prescription medication. However, to elucidate specific physiological effects of ST extracts, it is necessary to first understand which of its constituents are the major contributors to beneficial effects demonstrated for ST in this context. Aim of the studyTo determine an anxiolytic- and antidepressant-like effective concentration of a ST extract by means of a dose response in zebrafish (ZF) larvae, and to assess relative contributions of equivalent concentrations of isolated alkaloids contained in the effective concentration(s). Materials and methodsA dose response study employing a light-dark transition test (LDTT) was done in ZF larvae (<5 days post fertilization) to track locomotor activity in terms of anxiety-like (hyperlocomotion) and depression-like (hypolocomotion) behaviour. Larvae were treated for 1 h directly before the LDTT with escalating concentrations of a ST extract commercially known as Zembrin® (Zem) ranging from 0.25 to 500 μg/mL and compared to an untreated control group (n = 12 per treatment concentration). LDTT was repeated after 24 h to evaluate long-term exposure toxicity. The concentration that best attenuated hyperlocomotion during the dark phase following light-dark transition was identified as the anxiolytic-like concentration. This concentration, plus one higher and one lower concentration, were used for subsequent tests. The percentage content of each alkaloid (mesembrine, mesembrenone, mesembrenol, and mesembranol) in these concentrations were calculated and applied to additional larvae to identify the most effective anxiolytic-like alkaloid in the LDTT. To identify antidepressant-like therapeutic concentration and equivalent alkaloid concentration, the same treatment concentrations were tested in larvae (n = 12 per treatment concentration) pre-exposed to reserpine for 24 h. Depending on normality of data distribution, Brown-Forsythe and Welch, or Kruskal-Wallis ANOVA were used, with Dunnett or Dunn's multiple comparisons tests. ResultsOnly the extreme concentration of Zem (500 μg/mL) elicited toxicity after treatment for 24 h. Zem 12.5 μg/mL was the most effective anxiolytic-like concentration as it significantly decreased locomotor activity (P = 0.05) in the LDTT. Low (5 μg/mL), optimal (12.5 μg/mL) and high (25 μg/mL) Zem concentrations, as well as treatment solutions of single alkaloids (mesembrine, mesembrenone, mesembranol and mesembrenol), prepared to contain equivalent concentrations of each major alkaloid contained within these three concentrations of Zem, were tested further. Only mesembrine concentrations equal to that contained within the optimal and high dose of Zem (12.5 and 25 μg/mL) showed significant anxiolytic-like effects (P < 0.05). Only the highest Zem concentration (25 μg/mL) reversed the effects of reserpine - indicating antidepressant-like properties (P < 0.05) - while isolated alkaloids failed to induce such effects when administered in isolation. ConclusionsCurrent data provide evidence of both anxiolytic- and antidepressant-like effect of whole extract of Zem, with relatively higher concentrations required to achieve antidepressant-like effect. Of all alkaloids assessed, only mesembrine contributed significantly to the anxiolytic-like effects of Zem. No alkaloid alone could be pinpointed as a contributor to the antidepressant-like activity observed for higher concentration Zem. This may be due to synergistic effects of the alkaloids or may be due to other components not tested here. Current data warrants further investigation into mechanisms of action, as well as potential synergy, of ST alkaloids in suitable mammalian in vivo models.

Paraventricular nucleus-central amygdala oxytocinergic projection modulates pain-related anxiety-like behaviors in mice.

Anxiety disorders associated with pain are a common health problem. However, the underlying mechanisms remain poorly understood. We aimed to investigate the role of paraventricular nucleus (PVN)-central nucleus of the amygdala (CeA) oxytocinergic projections in anxiety-like behaviors induced by inflammatory pain. After inflammatory pain induction by complete Freund's adjuvant (CFA), mice underwent elevated plus maze, light-dark transition test, and marble burying test to examine the anxiety-like behaviors. Chemogenetic, optogenetic, and fiber photometry recordings were used to modulate and record the activity of the oxytocinergic projections of the PVN-CeA. The key results are as follows: inflammatory pain-induced anxiety-like behaviors in mice accompanied by decreased activity of PVN oxytocin neurons. Chemogenetic activation of PVN oxytocin neurons prevented pain-related anxiety-like behaviors, whereas inhibition of PVN oxytocin neurons induced anxiety-like behaviors in naïve mice. PVN oxytocin neurons projected directly to the CeA, and microinjection of oxytocin into the CeA blocked anxiety-like behaviors. Inflammatory pain also decreased the activity of CeA neurons, and optogenetic activation of PVNoxytocin -CeA circuit prevented anxiety-like behavior in response to inflammatory pain. The results of our study suggest that oxytocin has anti-anxiety effects and provide novel insights into the role of PVNoxytocin -CeA projections in the regulation of anxiety-like behaviors induced by inflammatory pain.

The prelimbic cortex but not the anterior cingulate cortex plays an important role in social recognition and social investigation in mice.

The prefrontal cortex (PFC) has been implicated in social cognitive functions and emotional behaviors in rodents. Each subregion (prelimbic cortex, PL; infralimbic cortex; and anterior cingulate cortex, ACC) of the PFC appears to play a different role in social and emotional behaviors. However, previous investigations have produced inconsistent data, and few previous studies directly compared the roles of the PFC subregions using the same experimental paradigm. Accordingly, in the present study, we examined the role of the PL and the ACC in short-term social recognition, social investigation, and anxiety-related behaviors in C57BL/6J mice. We subjected mice with a lesioned PL or ACC, as well as those in a sham control group, to tests of social recognition and social novelty where juvenile and adult male mice were used as social stimuli. In the social recognition test, the PL-lesioned mice exhibited habituation but not dishabituation regardless of whether they encountered juvenile or adult mice. In a subsequent social novelty test, they spent less time engaged in social investigation compared with the control mice when adult mice were used as social stimuli. These results suggest that PL lesions impaired both social recognition and social investigation. In contrast, ACC-lesioned mice did not exhibit impaired short-term social recognition or social investigation regardless of the social stimulus. Furthermore, PL lesions and ACC lesions did not affect anxiety-related behavior in the open field test or light-dark transition test. Our findings demonstrate that the PL but not the ACC plays an important role in social recognition and social investigation.

Electric foot-shock induces neurobehavioral aberrations due to imbalance in oxidative status, stress hormone, neurochemical profile, and irregular cortical-beta wave pattern in rats: A validated animal model of anxiety

Neuropsychiatric disorders can be modeled on animals to investigate the neural mechanism underlying these disorders. Models of neuropsychiatric disorders, such as anxiety, basically aim to produce the signs and symptoms of human anxiety disorders in laboratory animals. Electric foot-shock is recommended to induce anxiety-like symptoms in rodents. For this purpose, however, a range of current intensities is available in the literature. The present study aims to modify the existing practices of generating anxiety-like symptoms through electric foot-shock by identifying an optimum current intensity and combining it with behavioral paradigms to produce a rat model of anxiety. Furthermore, the validity of the model was confirmed by checking the fulfillment of three validity criteria necessary for the development of any disease model including face validity, construct validity, and predictive validity. In the current study, after pre-testing, 1.0 mA electric intensity was selected to produce the model of anxiety. The results showed that the induction of 1.0 mA electric foot-shock induces abnormal behavioral effects which were similar to anxiety-like effects as evident by social interaction test, light-dark transition test, and open field test. Moreover, aberrations in the levels of the stress hormone, oxidative stress parameters, hippocampal neurotransmitter levels, and cortical-EEG wave pattern were also observed in the rat model of anxiety which were successfully overcome using diazepam. In conclusion, the outcome of our study suggests that electric foot-shock can be an adequate stressor to produce a validated animal model of anxiety and this model can be confidently used to identify and screen new and/or novel anxiolytics.

Alleviation of Cocaine Withdrawal and Pertinent Interactions between Salvinorin-Based Antagonists and Kappa Opioid Receptor.

The kappa opioid receptor (KOR) is involved in the regulation of both the reward and mood processes. Recent reports find that the use of drugs of abuse increases the production of dynorphin and the overall activation of KOR. Long-acting KOR antagonists, such as norbinaltorphimine (nor-BNI), JDTic, and 5'-guanidinonaltrindole (GNTI), have been shown to stop depressive and anxiety-related disorders, which are the common side effects of withdrawal that can lead to a relapse in drug use. Unfortunately, these prototypical KOR antagonists are known to induce selective KOR antagonism that is delayed by hours and extremely prolonged, and their use in humans comes with serious safety concerns because they possess a large window for potential drug-drug interactions. Furthermore, their persistent pharmacodynamic activities can hinder the ability to reverse unanticipated side effects immediately. Herein, we report our studies of the lead selective, salvinorin-based KOR antagonist (1) as well as nor-BNI on C57BL/6N male mice for spontaneous cocaine withdrawal. Assessment of pharmacokinetics showed that 1 is a short-acting compound with an average half-life of 3.75 h across different compartments (brain, spinal cord, liver, and plasma). Both 1 (5 mg/kg) and nor-BNI (5 mg/kg) were shown to reduce spontaneous withdrawal behavior in mice, with 1 producing additional anti-anxiety-like behavior in a light-dark transition test (however, no mood-related effects of 1 or nor-BNI were observed at the current dosing in an elevated plus maze or a tail suspension test). Our results support the study of selective, short-acting KOR antagonists for the treatment of psychostimulant withdrawal and the associated negative mood states that contribute to relapse. Furthermore, we identified pertinent interactions between 1 and KOR via computational studies, including induced-fit docking, mutagenesis, and molecular dynamics simulations, to gain insight into the design of future selective, potent, and short-acting salvinorin-based KOR antagonists.

Osteocalcin ameliorates cognitive dysfunctions in a mouse model of Alzheimer’s Disease by reducing amyloid β burden and upregulating glycolysis in neuroglia

Alzheimer’s disease (AD) is the most common neurodegenerative disease characterized by the accumulation of amyloid β peptides (Aβ) and impaired glucose metabolism in the brain. Osteocalcin (OCN), an osteoblast-derived protein, has been shown to modulate brain functions but whether it has any effect on AD is undetermined. In this study, daily intraperitoneal injection of OCN for 4 weeks ameliorated the anxiety-like behaviors and cognitive dysfunctions in the APP/PS1 transgenic AD mice model, as shown in the increased entries into the central area in open field test, the increased time and entries into open arms in elevated plus maze test, the increased time spent in the light chamber in light-dark transition test, as well as the reduced escape latency and the increased preference for target quadrant in Morris water maze test. Aβ burden in the hippocampus and cortex of AD mice was ameliorated by OCN. Besides, OCN improved the neural network function of the brain, mainly in the enhanced power of high gamma band in the medial prefrontal cortex of AD mice. The proliferation of astrocytes in the hippocampus in AD mice was also inhibited by OCN as demonstrated by immunofluorescence. Furthermore, OCN enhanced glycolysis in astrocytes and microglia, as evidenced by elevated glucose consumption, lactate production, and increased extracellular acidification rate. Such an effect was abolished when the receptor of OCN – Gpr158 was knockdown in astrocytes. Our study revealed OCN as a novel therapeutic factor for AD potentially through reducing Aβ burden and upregulation of glycolysis in neuroglia.

Prefrontal cortex is necessary for long-term social recognition memory in mice

The prefrontal cortex (PFC) plays critical roles in social cognition and emotional regulation in humans and rodents; however, its involvement in social recognition memory in mice remains unclear. Here, we examined the roles of the PFC in short-term and long-term social recognition memory, social motivation, and anxiety-related behavior in C57BL/6J male mice. Sham control and PFC-lesioned mice underwent four different behavioral tests. In the social recognition test, composed of three daily trials over 3 consecutive days, the control mice spent less time investigating the juvenile stimulus mouse both within each day and across days. By contrast, while social investigation behavior in PFC-lesioned mice decreased across the three trials within each day, it did not decrease over the 3-day testing period. These results indicate that the PFC has an important role in long-term, but not short-term, social recognition memory. The control and PFC-lesioned mice exhibited similar social motivation in the three-chamber test – both groups preferred the juvenile mouse to the empty cylinder and did not prefer the adult mouse. In addition, the PFC lesion had no impact on anxiety-related behavior or general activity in the light–dark transition test or the open field test. Our findings demonstrate that the PFC is essential for long-term social recognition memory and that it plays a critical role in higher-order social cognition.

Effects of methylphenidate after a long period of discontinuation include changes in exploratory behavior and increases brain activities of Na+,K+-ATPase and acetylcholinesterase

Methylphenidate (MPH) has been widely misused by children and adolescents who do not meet all diagnostic criteria for attention-deficit/hyperactivity disorder. Since it is not yet known whether MPH can be administered in childhood without consequences in adulthood, in the present study we proposed to investigate the effects of chronic early treatment with MPH after a long period of discontinuation. Wistar male rats were injected with MPH (2 mg/kg, intraperitoneally) or saline solution once daily from 15th to 44th day of life. Two months after the last MPH administration, we evaluated the animal’s performances on a battery of behavior tests. We also tested Na+,K+-ATPase and acetylcholinesterase activities in prefrontal cortex and hippocampus, which may be associated with behavior. Rats treated with MPH during peri-adolescence show changes in exploratory behavior in adulthood in the open field but not in the elevated plus maze and light–dark transition tests. MPH-treated rats showed a lower latency to find the platform in the training phase, as well as a better performance in the test phase in the Morris water maze test. No differences were observed in the object recognition index and working memory. Acetylcholinesterase was increased in prefrontal cortex and hippocampus, while Na+,K+-ATPase was increased only in hippocampus. These findings provide additional evidence that early-life exposure to MPH can have complex effects in adulthood and new basis for understanding the behavioral and neurochemical consequences associated with chronic use of MPH during the development of central nervous system.

Zebrafish Larvae Rapidly Recover from Locomotor Effects and Neuromuscular Alterations Induced by Cholinergic Insecticides.

Owing to the importance of acetylcholine as a neurotransmitter, many insecticides target the cholinergic system. Across phyla, cholinergic signaling is essential for many neuro-developmental processes including axonal pathfinding and synaptogenesis. Consequently, early-life exposure to such insecticides can disturb these processes, resulting in an impaired nervous system. One test frequently used to assess developmental neurotoxicity is the zebrafish light-dark transition test, which measures larval locomotion as a response to light changes. However, it is only poorly understood which structural alterations cause insecticide-induced locomotion defects and how persistent these alterations are. Therefore, this study aimed to link locomotion defects with effects on neuromuscular structures, including motorneurons, synapses, and muscles, and to investigate the longevity of the effects. The cholinergic insecticides diazinon and dimethoate (organophosphates), methomyl and pirimicarb (carbamates), and imidacloprid and thiacloprid (neonicotinoids) were used to induce hypoactivity. Our analyses revealed that some insecticides did not alter any of the structures assessed, while others affected axon branching (methomyl, imidacloprid) or muscle integrity (methomyl, thiacloprid). The majority of effects, even structural, were reversible within 24 to 72 h. Overall, we find that both neurodevelopmental and non-neurodevelopmental effects of different longevity can account for the reduced locomotion. These findings provide unprecedented insights into the underpinnings of insecticide-induced hypoactivity.

Brain systems in cocaine abstinence-induced anxiety-like behavior in rodents: A review

Cocaine use disorder (CUD) is a significant public health issue that generates substantial personal, familial, and economic burdens. Still, there are no FDA-approved pharmacotherapies for CUD. Cocaine-dependent individuals report anxiety during withdrawal, and alleviation of anxiety and other negative affective states may be critical for maintaining drug abstinence. However, the neurobiological mechanisms underlying abstinence-related anxiety in humans or anxiety-like behavior in rodents are not fully understood. This review summarizes investigations regarding anxiety-like behavior in mice and rats undergoing cocaine abstinence, as assessed using four of the most common anxiety-related assays: the elevated plus (or its derivative, the elevated zero) maze, open field test, light-dark transition test, and defensive burying task. We first summarize available evidence that cocaine abstinence generates anxiety-like behavior that persists throughout protracted abstinence. Then, we examine investigations concerning neuropeptide, neurotransmitter, and neuromodulator systems in cocaine abstinence-induced anxiety-like behavior. Throughout, we discuss how differences in sex, rodent strain, cocaine dose and dosing strategy, and abstinence duration interact to generate anxiety-like behavior.

Road Runoff Characterization: Ecotoxicological Assessment Combined with (Non-)Target Screenings of Micropollutants for the Identification of Relevant Toxicants in the Dissolved Phase

Road runoff (RR) is an important vector of micropollutants towards groundwater and soils, threatening the environment and ecosystems. Through combined chemical and biological approaches, the purpose of this study was to get insights on specific toxicants present in RR from two sites differing by their traffic intensity and their toxicological risk assessment. Non-target screening was performed by HRMS on RR dissolved phase. Ecotoxicological risk was evaluated in a zebrafish embryos model and on rat liver mitochondrial respiratory chain. Specific HRMS fingerprints were obtained for each site, reflecting their respective traffic intensities. Several micropollutants, including 1,3-diphenylguanidine (DPG) and benzotriazole (BZT) were identified in greater concentrations at the high-traffic site. The origin of DPG was confirmed by analyzing HRMS fingerprints from shredded tires. RR samples from each site, DPG and BZT were of relatively low toxicity (no mortality) to zebrafish embryos, but all generated distinct and marked stress responses in the light–dark transition test, while DPG/BZT mixes abolished this effect. The moderate-traffic RR and DPG inhibited mitochondrial complex I. Our study highlights (i) the unpredictability of pollutants cocktail effect and (ii) the importance of a multi-approaches strategy to characterize environmental matrices, essential for their management at the source and optimization of depollution devices.

Getting more out of the zebrafish light dark transition test

In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.

Sotalol Treatment may Interfere With Retrieval, Expression, and/or Reconsolidation Processes Thus Disrupting Traumatic Memories in a Post-Traumatic Stress Disorder Mice Model.

The processes by which fear memory is encoded, consolidated, and re-consolidated are extremely complex and appear to require the release of stress hormones, especially adrenaline (AD). AD improves contextual fear memory, acting specifically on peripheral β2-adrenoceptors. Propranolol (peripheral and central β-adrenoceptor antagonist) treatment was shown to prevent post-traumatic stress disorder (PTSD) development and reduce its symptoms. However, propranolol has several side effects. Thus, we aimed to evaluate if sotalol (a peripheral β-adrenoceptor antagonist) treatment interferes with retrieval, expression, and/or reconsolidation of traumatic memories in a validated mice model that mimics the signs/symptoms of PTSD, thus intending to decrease them. Female mice were induced with PTSD following an established protocol. Sotalol (2.0 mg/kg) or vehicle were administered on days 2, 7, and 14. The percentage of freezing was calculated, and behavioral tests were carried out. Catecholamines in plasma were quantified by HPLC with electrochemical detection. Quantitative real-time polymerase chain reaction (qPCR) was used to evaluate mRNA expression of NR4A family genes in hippocampus. Following the submission of the animals to the same aversive context on days 2, 7, and 14, sotalol-treated mice exhibited significant less freezing behavior. In the elevated plus-maze test, the time spent and number of entries in the open arms, and total arm entries were increased in sotalol-treated mice. Also, the light-dark transition test revealed higher time spent, number of transitions to the light, and total number of transitions in sotalol-treated mice. Moreover, plasma AD was significantly decreased in sotalol-treated mice. On day 14, sotalol-treated mice exhibited a decrease in mRNA expression of Nr4a1 in the hippocampus. In conclusion, in PTSD mice model, sotalol appears to decrease traumatic memories and anxiety-like behavior, probably due to a decrease in peripheral adrenergic activity, which influences traumatic memories. The effects of sotalol upon re-exposure to the traumatic context may be consistent with interference in the retrieval, expression, and/or reconsolidation processes of contextual traumatic memory, resulting in a long-term reduction of PTSD symptoms and signs. The decreased Nr4a1 mRNA expression in the hippocampal formation may be crucial for these mice to develop diminished traumatic contextual memories after sotalol therapy in PTSD.

Clozapine modulation of zebrafish swimming behavior and gene expression as a case study to investigate effects of atypical drugs on aquatic organisms

Mental illnesses affect more than 150 million people in Europe and lead to an increasing consumption of neuroactive drugs during the last twenty years. The antipsychotic compound, clozapine, is one of the most used psychotropic drugs worldwide, with potentially negative consequences for the aquatic environment. Hence, the objectives of the study presented here were the quantification of clozapine induced changes in swimming behavior of exposed Danio rerio embryos and the elucidation of the molecular effects on the serotonergic and dopaminergic systems. Yolk-sac larvae were exposed to different concentrations (0.2 mg/L, 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L and 6.4 mg/L) of clozapine for 116 h post-fertilization, and changes in the swimming behavior of the larvae were assessed. Further, quantitative real-time PCR was performed to analyze the expression of selected genes. The qualitative evaluation of changes in the swimming behavior of D. rerio larvae revealed a significant decrease of the average swimming distance and velocity in the light-dark transition test, with more than a 36% reduction at the highest exposure concentration of 6.4 mg/L. Furthermore, the total larval body length was reduced at the highest concentration. An in-depth analysis based on expression of selected target genes of the serotonin (slc6a4a) and dopamine (drd2a) system showed an upregulation at a concentration of 1.6 mg/L and above. In addition, a lower increase in expression was detected for biomarkers of general stress (adra1a and cyp1a2). Our data show that exposure to clozapine during development inhibits swimming activity of zebrafish larvae, which could, in part, be due to disruption of the serotonin- and dopamine system.