Rodent Tests Research Articles

Animal Behavior in Psychedelic Research.

Psychedelic-assisted psychotherapy holds great promise in the treatment of mental health disorders. Research into 5-hydroxytryptamine 2A receptor (5-HT2AR) agonist psychedelic compounds has increased dramatically over the past two decades. In humans, these compounds produce drastic effects on consciousness, and their therapeutic potential relates to changes in the processing of emotional, social, and self-referential information. The use of animal behavior to study psychedelics is under debate, and this review provides a critical perspective on the translational value of animal behavior studies in psychedelic research. Acute activation of 5-HT2ARs produces head twitches and unique discriminative cues, disrupts sensorimotor gating, and stimulates motor activity while inhibiting exploration in rodents. The acute treatment with psychedelics shows discrepant results in conventional rodent tests of depression-like behaviors but generally induces anxiolytic-like effects and inhibits repetitive behavior in rodents. Psychedelics impair waiting impulsivity but show discrepant effects in other tests of cognitive function. Tests of social interaction also show conflicting results. Effects on measures of time perception depend on the experimental schedule. Lasting or delayed effects of psychedelics in rodent tests related to different behavioral domains appear to be rather sensitive to changes in experimental protocols. Studying the effects of psychedelics on animal behaviors of relevance to effects on psychiatric symptoms in humans, assessing lasting effects, publishing negative findings, and relating behaviors in rodents and humans to other more translatable readouts, such as neuroplastic changes, will improve the translational value of animal behavioral studies in psychedelic research. SIGNIFICANCE STATEMENT: Psychedelics like LSD and psilocybin have received immense interest as potential new treatments of psychiatric disorders. Psychedelics change high-order consciousness in humans, and there is debate about the use of animal behavior studies to investigate these compounds. This review provides an overview of the behavioral effects of 5-HT2AR agonist psychedelics in laboratory animals and discusses the translatability of the effects in animals to effects in humans. Possible ways to improve the utility of animal behavior in psychedelic research are discussed.

Role of Orbitofrontal Cortex and Differential Effects of Acute and Chronic Stress on Motor Impulsivity Measured With 1-Choice Serial Reaction Time Test in Male Rats.

Deficits in motor impulsivity, that is, the inability to inhibit a prepotent response, are frequently observed in psychiatric conditions. Several studies suggest that stress often correlates with higher impulsivity. Among the brain areas affected by stress, the orbitofrontal cortex (OFC) is notable because of its role in impulse control. OFC subregions with unique afferent and efferent circuitry play distinct roles in impulse control, yet it is not clear what OFC subregions are engaged during motor impulsivity tasks. In this study we used a rodent test of motor impulsivity, the 1-choice serial reaction time test, to explore activation of OFC subregions either during a well-learned motor impulsivity task or in a challenge task with a longer wait time that increases premature responding. We also examined the effects of acute inescapable stress, chronic intermittent cold stress and chronic unpredictable stress on motor impulsivity. Fos expression increased in the lateral OFC and agranular insular cortex during performance in both the mastered and challenge conditions. In the ventral OFC, Fos expression increased only during challenge, and within the medial OFC, Fos was not induced in either condition. Inescapable stress produced a transient effect on premature responses in the mastered task, whereas chronic intermittent cold stress and chronic unpredictable stress altered premature responses in both conditions in ways specific to each stressor. These results suggest that different OFC subregions have different roles in motor impulse control, and the effects of stress vary depending on the nature and duration of the stressor.

Blocking Studies to Evaluate Receptor-Specific Radioligand Binding in the CAM Model by PET and MR Imaging.

Simple SummaryIn the development of new targeted radiopharmaceuticals, it is mandatory to demonstrate their target-specific binding. Rodents are still primarily used for these experiments. With respect to the 3Rs principles, the demand for alternative methods to reduce the number of animal experiments is continuously increasing. In the present study, we investigated whether radiotracer uptake specificity can be evaluated by blocking studies in the CAM model. PET and MR imaging were used to visualize and quantify ligand accumulation. It was demonstrated that the CAM model could be used to evaluate the target-specific binding of a radiopharmaceutical. Due to intrinsic limitations of the CAM model, animal testing will still be required at more advanced stages of compound development. Still, the CAM model could significantly reduce the number of experiments through early compound pre-selection.Inhibition studies in small animals are the standard for evaluating the specificity of newly developed drugs, including radiopharmaceuticals. Recently, it has been reported that the tumor accumulation of radiotracers can be assessed in the chorioallantoic membrane (CAM) model with similar results to experiments in mice, such contributing to the 3Rs principles (reduction, replacement, and refinement). However, inhibition studies to prove receptor-specific binding have not yet been performed in the CAM model. Thus, in the present work, we analyzed the feasibility of inhibition studies in ovo by PET and MRI using the PSMA-specific ligand [18F]siPSMA-14 and the corresponding inhibitor 2-PMPA. A dose-dependent blockade of [18F]siPSMA-14 uptake was successfully demonstrated by pre-dosing with different inhibitor concentrations. Based on these data, we conclude that the CAM model is suitable for performing inhibition studies to detect receptor-specific binding. While in the later stages of development of novel radiopharmaceuticals, testing in rodents will still be necessary for biodistribution analysis, the CAM model is a promising alternative to mouse experiments in the early phases of compound evaluation. Thus, using the CAM model and PET and MR imaging for early pre-selection of promising radiolabeled compounds could significantly reduce the number of animal experiments.

The selective 5-HT 1A receptor agonist, NLX-112, overcomes tetrabenazine-induced catalepsy and depression-like behavior in the rat.

Tetrabenazine, a preferential inhibitor of the vesicular monoamine transporter type 2, depletes the brain monoamines dopamine, serotonin and norepinephrine. Tetrabenazine and deutetrabenazine (Austedo ®) are used to treat chorea associated with Huntington's disease. However, both compounds are known to aggravate Parkinsonism and depression observed in Huntington's disease patients. NLX-112 (a.k.a. befiradol/F13640) is a highly selective, potent and efficacious serotonin 5-HT 1A agonist. In animal models, it has robust efficacy in combating other iatrogenic motor disorders such as L-DOPA-induced dyskinesia and has marked antidepressant-like activity in rodent tests. In the present study, we investigated, in rats, the efficacy of NLX-112 to counteract tetrabenazine-induced catalepsy (a model of Parkinsonism) and tetrabenazine-induced potentiation of immobility in the forced swim test (FST, a model to detect antidepressant-like activity). The prototypical 5-HT 1A agonist, (±)8-OH-DPAT, and the 5-HT 1A partial agonist/dopamine D2 receptor blocker, buspirone, were used as comparators. Both NLX-112 and (±)8-OH-DPAT (0.16-2.5 mg/kg p.o. or s.c., respectively) abolished catalepsy induced by tetrabenazine (2 mg/kg i.p.). In comparison, buspirone (0.63-5.0 mg/kg p.o.) was ineffective and even tended to potentiate tetrabenazine-induced catalepsy at 0.63 mg/kg. In the FST, NLX-112 and (±)8-OH-DPAT (0.63 mg/kg) strongly reduced immobility when administered alone but also significantly opposed potentiation of immobility induced by tetrabenazine (1.5 mg/kg i.p.). Buspirone (0.63 and 2.5 mg/kg p.o.) had no effect by itself or against tetrabenazine. These results strongly suggest that selective and highly efficacious 5-HT 1A agonists, such as NLX-112, may be useful in combating tetrabenazine-induced Parkinsonism and/or depression in Huntington's disease patients.

Mepirapim, a Novel Synthetic Cannabinoid, Induces Addiction-Related Behaviors through Neurochemical Maladaptation in the Brain of Rodents.

Mepirapim is a synthetic cannabinoid that has recently been abused for recreational purposes. Although serious side effects have been reported from users, the dangerous pharmacological effects of Mepirapim have not been scientifically demonstrated. In this study, we investigated the addictive potential of Mepirapim through an intravenous self-administration test and a conditioned place preference test in rodents. Moreover, to determine whether the pharmacological effects of Mepirapim are mediated by cannabinoid receptors, we investigated whether Mepirapim treatment induces cannabinoid tetrad symptoms in mice. Lastly, to identify Mepirapim induced neurochemical maladaptation in the brains of mice, we performed microdialysis, western blots and neurotransmitter enzyme-linked immunosorbent assays. In the results, Mepirapim supported the maintenance of intravenous self-administration and the development of conditioned place preference. As a molecular mechanism of Mepirapim addiction, we identified a decrease in GABAeric signalling and an increase in dopaminergic signalling in the brain reward circuit. Finally, by confirming the Mepirapim-induced expression of cannabinoid tetrad symptoms, we confirmed that Mepirapim acts pharmacologically through cannabinoid receptor one. Taken together, we found that Mepirapim induces addiction-related behaviours through neurochemical maladaptation in the brain. On the basis of these findings, we propose the strict regulation of recreational abuse of Mepirapim.

CD4 T-cell depletion prevents Lassa fever associated hearing loss in the mouse model.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), which presents as a lethal hemorrhagic disease in severe cases. LASV-induced hearing loss in survivors is a huge socioeconomic burden, however, the mechanism(s) leading to hearing loss is unknown. In this study, we evaluate in a mouse LF model the auditory function using auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) to determine the mechanisms underlying LASV-induced hearing loss. In the process, we pioneered measures of ABR and DPOAE tests in rodents in biosafety level 4 (BSL-4) facilities. Our T cell depletion studies demonstrated that CD4 T-cells play an important role in LASV-induced hearing loss, while CD8 T-cells are critical for the pathogenicity in the acute phase of LASV infection. Results presented in this study may help to develop future countermeasures against acute disease and LASV-induced hearing loss.

P599. The Muscarinic Agonist Xanomeline Demonstrates Standalone Activity and Augments Clinical Antipsychotics in Rodent Behavioral Models of Psychosis

Evidence indicates that muscarinic acetylcholine receptor agonists exhibit antipsychotic properties. Xanomeline, a centrally active M1 and M4 receptor agonist, is devoid of any direct dopamine receptor affinity or activity. An important question is whether xanomeline can also augment the effects of existing antipsychotics. Two rodent behavioral models were used to test combinations of xanomeline and either risperidone or aripiprazole. The conditioned avoidance response (CAR) and psychostimulant-induced locomotor activity (LMA) assays are commonly used behavioral tests in rodents that have good predictive validity for detecting antipsychotic activity.

The Effects of Social vs. Individual Housing of Zebrafish on Whole-Body Cortisol and Behavior in Two Tests of Anxiety.

Two of the most used models of anxiety in zebrafish research, the novel tank-diving test (NTDT) and the black-white preference test (BWPT), are modifications of assays used in rodent research (open field test and light/dark test). There has been a thorough validation of these tests in rodents, but a similar level of knowledge is still missing in zebrafish. Adult zebrafish naturally live in shoals with conspecifics, and group housing is therefore assumed to be the optimal housing condition for zebrafish, as it allows for shoaling behavior. This study investigated how housing in social isolation affected whole-body cortisol and the behavioral responses in the NTDT and BWPT. We also examined the correlation between the behavioral responses in the two behavioral tests. We found that zebrafish housed in groups had significantly higher whole-body cortisol than individually housed zebrafish (F1, 85 = 25.51, P < 0.0001). Regardless of treatment, all groups had a general preference for the lower compartment in the NTDT and the black compartment in the BWPT. Individually housed zebrafish had a higher total number of entries to the white compartment in BWPT compared to group housed zebrafish when their first test was BWPT (F1, 48 = 5.79, P = 0.0201), but not when BWPT was their second behavioral test. Fish that had higher whole-body cortisol had a tendency toward fewer entries into the white compartment the first 3 min of the BWPT (F1, 48 = 3.90, P = 0.0540). There was no effect of housing on the behaviors registered in the NTDT. There was a positive correlation (correlation coefficient 0.40; p = 0.003) between transitions from black to white compartment in BWPT and transitions from lower to upper compartment in NTDT, but we did not find any association between duration in white compartment in BWPT and upper compartment in NTDT. Considering this, we suggest that further model validation is needed.

Commonly-used rodent tests of anxiety-like behavior lack predictive validity for human sex differences

Women are more likely to develop an anxiety disorder than men. Yet, preclinical models of anxiety were largely developed in male rodents, with poorly understood predictive validity for sex differences. Here, we investigate whether commonly-used anxiety-like behavior tests, elevated plus maze (EPM) and open field (OF), represent the human sex difference in adult Sprague-Dawley rats. When interpreted by EPM or OF, female rats displayed less anxiety-like behavior compared to males, as they spent twice as much time in the open arms of the EPM or the center of the OF compared to males. However, they also displayed vastly different levels of locomotor activity, possibly confounding interpretation of these locomotion-dependent tests. To exclude locomotion from the assessment, the acoustic startle response (ASR) test was used. When interpreted by the ASR test, females displayed more anxiety-like behavior compared to males, as indicated by a nearly two-fold higher startle amplitude. The observed sex differences were not driven by gonadal steroids. Overall, all but one of the tests fail to mirror the sex difference in anxiety reported in humans. Our findings suggest that the ASR might be a better fit in modelling female anxiety-like behavior.

Comparative Studies on Dispersion Characters of Titanium Dioxide (TiO2) Nanoparticles for Rodent Toxicology Studies

This research aimed to find a suitable vehicle (media) for dispersing titanium dioxide nanoparticles (TiO2 NPs) to investigate their toxicity in laboratory rodents.The TiO2 NPs were suspended in different vehicles viz., distilled water, Tween 80 (0.1% v/v), normal saline, normal saline + Tween 80 (0.1% v/v) and carboxymethylcellulose (CMC; 0.1%, 0.2%, 0.3% or 0.4% w/v). The hydrodynamic diameters, zeta potential, and polydispersity index were measured by the dynamic light scattering (DLS) technique by employing a zeta sizer. The TiO2 NPs in Tween 80 (0.1% v/v) and CMC (0.2% w/v) showed lesser hydrodynamic diameter compared to other vehicles. Further, TiO2 NPs dispersed in CMC (0.1%, 0.2%, or 0.3% w/v) showed relatively better zeta potential in terms of better stability than other media examined. The polydispersity index of nanoparticles was relatively less for CMC: 0.1% w/v followed by CMC: 0.2% w/v. Thus, it is concluded that CMC (0.2% w/v) can be used as an ideal vehicle among the various media examined for delivery of TiO2 NPs for toxicity testing in laboratory rodents.

Intracerebroventricular infusion of the selective orexin 1 receptor antagonist SB-334867 impairs cognitive flexibility in a sex-dependent manner

Orexin neuropeptides are well known for their role in sleep/wake cycle, feeding behavior and motivation-related behaviors. However, their role in cognition is not clearly understood. We recently published that orexin deficiency impairs intra-dimensional set shifting in female homozygous orexin-deficient mice but improves the first reversal phase in male homozygous orexin-deficient mice in the attentional set shifting task (ASST), a well-established rodent test for cognitive flexibility. In the present study, we tested if intracerebroventricular injections of the selective orexin 1 receptor antagonist SB-334867 (4 µg/2 µL) affects cognitive flexibility in the different phases of ASST. We found that SB-334867 injections impaired the first and second reversal phases in female C57BL/6J mice but not in males. In addition, we also showed that at this particular dose of SB-334867, the consumption of the reward that was used in the ASST was not affected in both males and females. Our findings indicate that cognitive flexibility is impaired by orexin 1 receptor antagonism in a sex-dependent manner and reiterates the sexually dimorphic role of orexin in cognitive flexibility.

Lack of general learning ability factor in a rat test battery measuring a wide spectrum of cognitive domains.

Objective: In the framework of a larger project aiming to test putative cognitive enhancer drugs in a system with improved translational validity, we established a rodent test battery, where different, clinically relevant cognitive domains were investigated in the same animal population. The aim of the current study was to check whether performances in the different tasks representing different cognitive functions are assay-specific or may originate in an underlying general learning ability factor. Methods: In the experiments 36 Long-Evans and 36 Lister Hooded rats were used. The test battery covered the following cognitive domains: attention and impulsivity (measured in the 5-choice serial reaction time task), spatial memory (Morris water-maze), social cognition (cooperation task), cognitive flexibility (attentional set shifting test), recognition memory (novel object recognition) and episodic memory (water-maze based assay). The outcome variables were analyzed by correlation analysis and principal component analysis (PCA). The datasets consisted of variables measuring learning speed and performance in the paradigms. From the raw variables composite variables were created for each assay, then from these variables a composite score was calculated describing the overall performance of each individual in the test battery. Results: Correlations were only found among the raw variables characterizing the same assay but not among variables belonging to different tests or among the composite variables. The PCAs did not reduce the dimensionality of the raw or composite datasets. Graphical analysis showed variable performance of the animals in the applied tests. Conclusions: The results suggests the assay outcomes (learning performance) in the system are based on independent cognitive domains.

Influences of Taurine Pharmacodynamics and Sex on Active Avoidance Learning and Memory.

Researchers have begun to direct their research to focus on the use of taurine as a psychopharmacotherapeutic compound to treat a wide range of health- related conditions as well as neuropathological diseases. Moreover, taurine has been shown to improve emotional and cognitive declines associated with senescence in neurotypical animal models. However, despite these advances in the field of taurine therapeutics, much less is known regarding the effects of sex and taurine on neurotypical animal models that are then manipulated, modified, and/or mutated to study human diseases. The present study sought to investigate this matter in a Long Evans Hooded rat model of mature age (i.e., postnatal day 60-90) in an active avoidance test (AAT). Rats were trained for 20 trials, given a 1h. test break, retrained for another 20 trials, and then tested at 24h, 48h, and 1week for learning and memory retention. An N=63 rats were randomly assigned to three groups: (1) Control (n=22), (2) Taurine Pre-Train (n=19), and (3) Taurine Post-Train (n=20). The aim of the present study was to determine the effects of taurine given 15 min before training when compared to being given after training but 15 min before testing at 24h on learning and memory consolidation of the AAT. The results showed in Control rats that females had shorter latencies to cross in the shuttle box, increased rates of correct learning by the % Avoids/Escapes, and decreased rates of learning errors by the % Shocks. In Taurine Post-Train male rats, taurine treatment decreased their latency to cross in the shuttle box and their rate of learning errors by the % Shocks at 24h and 48h Testing, but it had no effect on their rate of correct learning by the % Avoids/Escapes when compared to Control and Taurine Pre-Train male rats. In contrast, Taurine Post-Train female rats increased their latency to cross in the shuttle box during Training, 24h and 48h Testing, when compared to the Control and Taurine Pre-Train female rats. Further, Taurine Post-Train female rats decreased their rate of learning % Avoids/Escapes and increased the rate of learning errors % Shocks when compared to Control female rats during Training and 24h Testing but decreased their rate of learning % Avoids/Escapes and increased the rate of learning errors % Shocks when compared to Taurine Pre-Train female rats across all test conditions. These findings suggest that neurotypical female rats may be more sensitive to the aversive stimuli (i.e., foot shocks) used in the AAT as a motivating factor for learning that may cause paradoxical behavioral learning and memory patterns. This phenomenon raises an important concern for researchers to consider when studying learning and behavioral tests in rodents that use aversive and non-aversive stimuli or a combination of both such as in the AAT. Taurine, albeit neuroprotective, may not have as much benefit in a neurotypical animal model and may increase the susceptibility for anxiogenic behaviors and interfere with cognitive learning and memory behaviors. Therefore, the mechanistic way(s) in which taurine can treat, recovery, ameliorate, and forestall other neuropathological diseases in animal models may have different psychopharmacodynamics and psychopharmacokinetics in a neurotypical animal model and should be studied with caution. This does not preclude the continued investigation of taurine psychopharmacotherapies for neuropathological diseases but encourages the careful investigation of taurine supplementation and treatment in neurotypical animals as paradoxical behavioral and cognitive outcomes have been observed herein.

Time to Abolish the Forced Swim Test in Rats for Depression Research?

Abstract The forced swim test (FST) is a controversial rodent test that has been used for decades, mainly in depression studies. The severity of the procedure makes it ethically questionable and its validity has also been questioned. In this paper we contribute new data to this debate. We identified original research papers related to Major Depressive Disorder (MDD), using rats as models. We compared the citations received by studies that used the FST and by studies that did not, within subsequent human medical papers. The results show that the number of citations received by both groups was very low, but in the papers describing the FST data the median citation number was zero. Citation analysis indicates that the FST is not contributing significantly to the understanding or cure of MDD. We briefly review other approaches that overcome the ethical limitations of the FST, and which might also surpass its efficacy.

A Preclinical Model of Computerized Cognitive Training: Touchscreen Cognitive Testing Enhances Cognition and Hippocampal Cellular Plasticity in Wildtype and Alzheimer's Disease Mice.

With the growing popularity of touchscreen cognitive testing in rodents, it is imperative to understand the fundamental effects exposure to this paradigm can have on the animals involved. In this study, we set out to assess hippocampal-dependant learning in the APP/PS1 mouse model of Alzheimer’s disease (AD) on two highly translatable touchscreen tasks – the Paired Associate Learning (PAL) task and the Trial Unique Non-Matching to Location (TUNL) task. Both of these tests are based on human tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and are sensitive to deficits in both mild cognitive impairment (MCI) and AD. Mice were assessed for deficits in PAL at 9–12 months of age, then on TUNL at 8–11 and 13–16 months. No cognitive deficits were evident in APP/PS1 mice at any age, contrary to previous reports using maze-based learning and memory tasks. We hypothesized that daily and long-term touchscreen training may have inadvertently acted as a cognitive enhancer. When touchscreen-tested mice were assessed on the Morris water maze, they showed improved task acquisition compared to naïve APP/PS1 mice and wild-type (WT) littermate controls. In addition, we show that touchscreen-trained WT and APP/PS1 mice show increased cell proliferation and immature neuron numbers in the dentate gyrus compared to behaviorally naïve WT and APP/PS1 mice. This result indicates that the touchscreen testing paradigm could improve cognitive performance, and/or mask an impairment, in experimental mouse models. This touchscreen-induced cognitive enhancement may involve increased neurogenesis, and possibly other forms of cellular plasticity. This is the first study to show increased numbers of proliferating cells and immature neurons in the hippocampus following touchscreen testing, and that touchscreen training can improve cognitive performance in maze-based spatial navigation tasks. This potential for touchscreen testing to induce cognitive enhancement, or other phenotypic shifts, in preclinical models should be considered in study design. Furthermore, touchscreen-mediated cognitive enhancement could have therapeutic implications for cognitive disorders.

Cognitive profiling and proteomic analysis of the modafinil analogue S-CE-123 in experienced aged rats

The lack of novel cognitive enhancer drugs in the clinic highlights the prediction problems of animal assays. The objective of the current study was to test a putative cognitive enhancer in a rodent cognitive test system with improved translational validity and clinical predictivity. Cognitive profiling was complemented with post mortem proteomic analysis. Twenty-seven male Lister Hooded rats (26 months old) having learned several cognitive tasks were subchronically treated with S-CE-123 (CE-123) in a randomized blind experiment. Rats were sacrificed after the last behavioural procedure and plasma and brains were collected. A label-free quantification approach was used to characterize proteomic changes in the synaptosomal fraction of the prefrontal cortex. CE-123 markedly enhanced motivation which resulted in superior performance in a new-to-learn operant discrimination task and in a cooperation assay of social cognition, and mildly increased impulsivity. The compound did not affect attention, spatial and motor learning. Proteomic quantification revealed 182 protein groups significantly different between treatment groups containing several proteins associated with aging and neurodegeneration. Bioinformatic analysis showed the most relevant clusters delineating synaptic vesicle recycling, synapse organisation and antioxidant activity. The cognitive profile of CE-123 mapped by the test system resembles that of modafinil in the clinic showing the translational validity of the test system. The findings of modulated synaptic systems are paralleling behavioral results and are in line with previous evidence for the role of altered synaptosomal protein groups in mechanisms of cognitive function.

Analixity: An open source, low-cost analysis system for the elevated plus maze test, based on computer vision techniques

Manual analysis of behavioral tests in rodents involves inspection of video recordings by a researcher that assesses rodent movements to quantify parameters related with a behavior of interest. The assessment of the researcher during the quantification of such parameters can introduce variability among experimental conditions or among sessions of analysis. Here, we introduce Analixity, a video processing software for the elevated plus maze test (EPM), in which quantification of behavioral parameters is automatic, reducing the time spent in analysis and solving the variability problem. Analixity is an adaptable multiplatform open-source system. Analixity generates an Excel file with the quantified behavioral variables, such as time spent in open and closed arms and in the center zone, number of entries to each zone and total distance traveled during the test. For validation, we compared results obtained by Analixity with results obtained by manual analysis. We did not find statistically significant differences. In addition, we compared the results obtained by Analixity with results obtained by the commercial software ANY-maze. We did not find statistically significant differences in the quantification of parameters such as time spent in open arms, time spent in closed arms, time spent in center zone, number of closed arms, open arms entries, and anxiety index. We concluded that Analixity is an open-source software as reliable and effective as a commercial software.

Employing an adverse outcome pathway framework for weight-of-evidence assessment with application to the ICH S1B guidance addendum

Across industry, there is a paradigm shift occurring for carcinogenicity testing, with the focus moving from long term animal studies to alternative approaches. Based on the explorative work done in recent years, the International Council for Harmonization (ICH) recently published a draft addendum to the S1B guidance, which allows for a weight-of-evidence (WoE) assessment to be conducted based on data gathered throughout the pharmaceutical development process and literature to mitigate some testing in rodents if the body of evidence clearly shows undertaking an animal lifetime study would not add value to the risk assessment. While several alternative approaches already exist, and other new approach methodologies (NAMs) are being explored, all of which can contribute to this WoE, it is important that all the evidence can be combined in a meaningful and consistent way to reach a conclusion. Adverse outcome pathways have been advocated as a framework for organising evidence in an integrated approach to testing and assessment, which gives context to data and can aid reaching a conclusion as to the adverse outcome (AO). This approach can be combined with a reasoning methodology to give a prediction for an AO and applied to the factors which need to be considered for the ICH S1B WoE to predict for carcinogenicity. Using this approach to the WoE assessment, consistent, scientifically robust, and transparent calls can be made as to whether conducting an animal carcinogenicity study would add value to a human risk assessment and mitigate the need to run animal studies unnecessarily.

Anti-nociceptive effects of ECa 233 a standardized extract of Centella asiatica (L.) Urban on chronic neuropathic orofacial pain in mice

Ethnopharmacological relevanceECa 233 is a standardized extract of Centella asiatica (L.) Urban, a herb traditionally used to treat a number of diseases including neurological disorders. Accordingly, ECa 233 showed benefits on animal models of cognitive deficits, chronic stress and Parkinson's disease. Analgesic activity of ECa 233 was shown in Tail's flick test in rodent and relieving aphthous ulcer pain in man. Moreover, acute and sub-chronic toxicity testing in rodents and pharmacokinetic study in healthy volunteers, clinical trial phase I demonstrated good safety profiles of ECa 233. Aim of the studyThis study aims to evaluate the anti-nociceptive effects of ECa 233 and its synergistic effect with gabapentin on chronic neuropathic orofacial pain after 3 weeks infraorbital nerve chronic constriction injury in mice. The peripheral and central nociceptive activities are also examined. Materials and methodsChronic neuropathic orofacial pain was induced by 3 weeks infraorbital nerve chronic constriction injury. Mice were treated with ECa 233 (30, 100 and 300 mg/kg) and gabapentin (10 mg/kg) by oral gavage starting on day 21 and going on for 14 consecutive days. Mechanical hyperalgesia and allodynia were measured on day 7, 14, 21, 28 and 35 after infraorbital nerve chronic constriction injury. At the end of the experiment, mice were observed for the sedative effect using the locomotor activity, the calcitonin gen-related peptide in trigeminal ganglion and c-fos expression in trigeminal nucleus caudalis were investigated after euthanasia. ResultsInfraorbital nerve chronic constriction injury gradually induced marked ipsilateral mechanical hyperalgesia and allodynia. The maximum hyperalgesia and allodynia response presented on day 21 and the response was remained constant until day 35. Treatment with either 300 mg/kg ECa 233 or 10 mg/kg gabapentin were able to attenuate mechanical hyperalgesia and allodynia. The downregulation of calcitonin gen-related peptide on ipsilateral trigeminal ganglion were observed in ECa 233 at 100 and 300 mg/kg and 10 mg/kg gabapentin-treated groups. The c-fos expression on ipsilateral trigeminal nucleus caudalis was also decreased in 300 mg/kg ECa 233 and 10 mg/kg gabapentin-treated groups. ConclusionECa 233 reduced hyperalgesia and allodynia by modulating the peripheral calcitonin gen-related peptide expression consequently alleviate the nociceptive activity in trigeminal nucleus caudalis. Further clinical trial to proof ECa 233's efficacy in neuropathic pain in man as well as possible attributable mechanism of action should be further investigated.

Effect of Estrous Cycle on Behavior of Females in Rodent Tests of Anxiety.

Anxiety disorders are more prevalent in women than in men. In women the menstrual cycle introduces another variable; indeed, some conditions e.g., premenstrual syndrome, are menstrual cycle specific. Animal models of fear and anxiety, which form the basis for research into drug treatments, have been developed almost exclusively, using males. There remains a paucity of work using females and the available literature presents a confusing picture. One confound is the estrous cycle in females, which some authors consider, but many do not. Importantly, there are no accepted standardized criteria for defining cycle phase, which is important given the rapidly changing hormonal profile during the 4-day cycle of rodents. Moreover, since many behavioral tests that involve a learning component or that consider extinction of a previously acquired association require several days to complete; the outcome may depend on the phase of the cycle on the days of training as well as on test days. In this article we consider responsiveness of females compared to males in a number of commonly used behavioral tests of anxiety and fear that were developed in male rodents. We conclude that females perform in a qualitatively similar manner to males in most tests although there may be sex and strain differences in sensitivity. Tests based on unconditioned threatening stimuli are significantly influenced by estrous cycle phase with animals displaying increased responsiveness in the late diestrus phase of the cycle (similar to the premenstrual phase in women). Tests that utilize conditioned fear paradigms, which involve a learning component appear to be less impacted by the estrous cycle although sex and cycle-related differences in responding can still be detected. Ethologically-relevant tests appear to have more translational value in females. However, even when sex differences in behavior are not detected, the same outward behavioral response may be mediated by different brain mechanisms. In order to progress basic research in the field of female psychiatry and psychopharmacology, there is a pressing need to validate and standardize experimental protocols for using female animal models of anxiety-related states.