Activation of NOS-cGMP pathways promotes stress-induced sensitization of behavioral responses in zebrafish.

Nitric oxide (NO) is a molecule involved in plasticity across levels and systems. The role of NOergic pathways in stress-induced sensitization (SIS) of behavioral responses, in which a particular stressor triggers a state of hyper-responsiveness to other stressors after an incubation period, was assessed in adult zebrafish. In this model, adult zebrafish acutely exposed to a fear-inducing conspecific alarm substance (CAS) and left undisturbed for an incubation period show increased anxiety-like behavior 24 h after exposure. CAS increased forebrain glutamate immediately after stress and 30 min after stress, an effect that was accompanied by increased nitrite levels immediately after stress, 30 min after stress, 90 min after stress, and 24 h after stress. CAS also increased nitrite levels in the head kidney, where cortisol is produced in zebrafish. CAS-elicited nitrite responses in the forebrain 90 min (but not 30 min) after stress were prevented by a NOS-2 blocker. Blocking NOS-1 30 min after stress prevents SIS; blocking NOS-2 90 min after stress also prevents stress-induced sensitization, as does blocking calcium-activated potassium channels in this latter time window. Stress-induced sensitization is also prevented by blocking guanylate cyclase activation in both time windows, and cGMP-dependent channel activation in the second time window. These results suggest that different NO-related pathways converge at different time windows of the incubation period to induce stress-induced sensitization.

Gametogenesis and seminatural reproduction of the Amazon twospot astyanax Astyanax bimaculatus (Linnaeus, 1758) cultivated in an enriched environment.

Environmental enrichment is used to provide well-being to the animals, such as fish, in captive conditions, mimicking their natural habitat. It may influence fish behavior, physiology, and survival. In terms of reproduction, however, the relationship between environment enrichment and successful reproduction in captivity is still poorly explored in fish species. Aiming to understand any possible benefits of structural enrichment on fish reproduction, 10-days-hatched larvae of the twospot astyanax Astyanax bimaculatus were raised for 18 weeks in tanks with different elements of structural environmental enrichment (PVC pipes, stones, and artificial plants). In the 5th month of life, those animals were hormonally induced to reproduce to assess gamete formation and offspring quality. Animals raised in a sterile-reared environment (non-enriched) showed earlier spawning than the enriched one, presenting significant quantities of Postovulatory follicle complexes (POCs) and cells in atresia in female ovaries, indicating possible reproductive dysfunction or stress, as well as a greater quantity of empty testicular lumen in males, indicating great release of sperm. On the contrary, animals cultivated in enriched environments showed gonads filled with semen in males and vitellogenic oocytes in females. Furthermore, offspring from the sterile-reared group presented significant rates of larval abnormality compared to the enriched group. In conclusion, the results of this study show that environmental enrichment can interfere with the reproduction of fish in captivity, mainly by preventing early maturation of gametes, which can result in low-quality offspring and, consequently, low production of fish species.

Fish personality: meta-theoretical issues, personality dimensions, and applications to neuroscience and psychopathology.

While the field of personality neuroscience has extensively focused on humans and, in a few cases, primates and rodents, a wide range of research on fish personality has emerged in the last decades. This research is focused mainly on the ecological and evolutionary causes of individual differences and also aimed less extensively at proximal mechanisms (e.g., neurochemistry or genetics). We argue that, if consistent and intentional work is made to solve some of the meta-theoretical issues of personality research both on fish and mammals, fish personality research can lead to important advances in personality neuroscience as a whole. The five dimensions of personality in fish (shyness-boldness, exploration-avoidance, activity, aggressiveness, and sociability) need to be translated into models that explicitly recognize the impacts of personality in psychopathology, synergizing research on fish as model organisms in experimental psychopathology, personality neuroscience, and ecological-ethological approaches to the evolutionary underpinnings of personality to produce a powerful framework to understand individual differences.

Social investigation and social novelty in zebrafish: Roles of salience and novelty.

Social preference tests can be used to analyze variables that influence and modify social behaviors, and to investigate effects of substances such as medications, drugs, and hormones. They may become important tools for finding a valid model to study neuropsychiatric changes and to study human neurodevelopmental processes that have been impaired by social events. While a preference for conspecifics has been shown for different species, social novelty has been used as a model for anxiety-like behavior in rodents. The goal of this research was to understand the roles of stimulus salience (numerousness) and novelty in social investigation and social novelty tests in zebrafish (Danio rerio Hamilton 1822). We used a sequential design, in which animals are exposed first to a social investigation test (with dichotomous presentation of novel conspecifics vs. empty tank) and then to a social novelty test (with dichotomous presentation of the already known conspecific and a novel conspecific). In experiment 1, animals were presented to either 1 or 3 (vs. an empty tank) conspecifics as stimuli. In experiment 2, animals were presented to 1 vs. 3 conspecifics as stimuli. In experiment 3, animals were observed in the social investigation and social novelty tests for 3 consecutive days. The results showed equivalence between 1 or 3 conspecifics in the social investigation and social novelty tests, although animals were able to discriminate between different shoal sizes. These preferences do not change with repeated test exposure, suggesting novelty to be a minor contributor to social investigation and social novelty in zebrafish.

Oogenesis and in vitro reproduction of the twospot astyanax Astyanax bimaculatus (Linnaeus, 1758) exposed to conspecific alarm substance.

Stress situations can be essential to trigger reproduction in fish; however, it may also inhibit it. One of those situations involves the release of the conspecific alarm substance (CAS), a natural stressor, into the water by specific fish epidermal cells after a predator attack. Little is known about the effects of that substance on fish reproduction. This study aimed to evaluate the effects of CAS exposure on the oogenesis and reproduction of the twospot astyanax Astyanax bimaculatus before the hormonal induction for artificial reproduction. No macroscopic or cellular changes in the ovaries were observed for the females exposed to CAS, and the oocyte stages show all females in the same phase of maturation (Spawning Capable). Females exposed to CAS spawned 20 min before the females without exposure. On the other hand, they ovulated only once, whereas the females from the control group ovulated multiple times for approximately two hours after hormonal induction. Moreover, the precocious ovulation of the females submitted to CAS did not generate offspring, since all generated zygotes did not develop. In contrast, the control group females produced more than 11 thousand healthy larvae. Exposing the female fish to CAS during their reproductive management in captivity may reduce breeding success.

Biocultural psychopathology as a new epistemology for mental disorders.

Psychopathology has been criticized for decades for its reliance on a brain-centred and over-reductionist approach which views mental disorders as disease-like natural kinds. While criticisms of brain-centred psychopathologies abound, these criticisms sometimes ignore important advances in the neurosciences which view the brain as embodied, embedded, extended and enactive, and as fundamentally plastic. A new onto-epistemology for mental disorders is proposed, focusing on a biocultural model, in which human brains are understood as embodied and embedded in ecosocial niches, and with which individuals enact particular transactions characterized by circular causality. In this approach, neurobiological bases are inseparable from interpersonal and socio-cultural factors. This approach leads to methodological changes in how mental disorders are studied and dealt with.

Roles of the 5-HT2C receptor on zebrafish sociality.

Serotonin (5-HT) receptors have been implicated in social behavior in vertebrates. Zebrafish (Danio rerio) have been increasingly being used behavioral neuroscience to study the neurobiological correlates of behavior, including sociality. Nonetheless, the role of 5-HT2C receptors in different social functions were not yet studied in this species. Zebrafish were treated with the agonist MK-212 (2 mg/kg) or the antagonist RS-102221 (2 mg/kg) and tested in the social interaction and social novelty tests, conditional approach test, or mirror-induced aggressive displays. MK-212 increased preference for an unknown conspecific in the social investigation test, but also increased preference for the known conspecific in the social novelty test; RS-102221, on the other hand, decreased preference in the social investigation test but increased preference for the novel conspecific in the social novelty test. MK-212 also decreased predator inspection in the conditional approach test. While RS-102221 decreased time in the display zone in the mirror-induced aggressive display test, it increased display duration. Overall, these results demonstrate the complex role of 5-HT2C receptors in different social contexts in zebrafish, revealing a participation in social plasticity in vertebrates.

5-HT2C agonists and antagonists block different components of behavioral responses to potential, distal, and proximal threat in zebrafish.

Serotonin (5-HT) receptors have been implicated in responses to aversive stimuli in mammals and fish, but its precise role is still unknown. Moreover, since at least seven families of 5-HT receptors exist in vertebrates, the role of specific receptors is still debated. Aversive stimuli can be classified as indicators of proximal, distal, or potential threat, initiating responses that are appropriate for each of these threat levels. Responses to potential threat usually involve cautious exploration and increased alertness, while responses to distal and proximal threat involve a fight-flight-freeze reaction. We exposed adult zebrafish to a conspecific alarm substance (CAS) and observed behavior during (distal threat) and after (potential threat) exposure, and treated with the 5-HT2C receptor agonists MK-212 or WAY-161503 or with the antagonist RS-102221. The agonists blocked CAS-elicited defensive behavior (distal threat), but not post-exposure increases in defensive behavior (potential threat), suggesting inhibition of responses to distal threat. MK-212 blocked changes in freezing elicited by acute restraint stress, a model of proximal threat, while RS-102221 blocked changes in geotaxis elicited this stressor. We also found that RS-102221, a 5-HT2C receptor antagonist, produced small effect on behavior during and after exposure to CAS. Preprint: https://www.biorxiv.org/content/10.1101/2020.10.04.324202; Data and scripts: https://github.com/lanec-unifesspa/5-HT-CAS/tree/master/data/5HT2C.

Open Practical Laboratories in the Neurosciences: An outreach program for neuroscience communication in middle schools.

The Open Practical Laboratory in the Neurosciences is an outreach program that seeks to improve the knowledge of the neurosciences by elementary school students and to promote better attitudes in relation to neuroscience, science in general, and scientists. It consists in practical and demonstration activities on the theme of the neurosciences. This outreach strategy was applied in four public schools in a municipality in Southeastern Pará, Brazil characterized by low performance in educational reviews in Brazil. Students from both sexes from 9th grade (last grade before high school) were enrolled in the program. The outreach program improved students' knowledge about the neurosciences, and promoted better attitudes in relation to science and scientists, in comparison to students who did not go through the activities. Results suggest that scientific outreach strategies can produce results beyond immediate increases in knowledge, promoting better attitudes in relation to science.

Embryonic development of the fire-eye-tetra Moenkhausia oligolepis (Characiformes: Characidae).

This study describes the embryonic development of Moenkhausia oligolepis in laboratory conditions. After fertilization, the embryos were collected every 10 min up to 2 h, then every 20 min up to 4 h, and afterwards every 30 min until hatching. The fertilized eggs of M. oligolepis measured approximately 0.85 ± 0.5 mm and had an adhesive surface. Embryonic development lasted 14 h at 25ºC through the zygote, cleavage, blastula, gastrula, neurula, and segmentation phases. Hatching occurred in embryos around the 30-somites stage. The present results contribute only the second description of embryonic development to a species from the Moenkhausia genus, being also the first for this species. Such data are of paramount importance considering the current conflicting state of this genus phylogenetic classification and may help taxonomic studies. Understanding the biology of a species that is easily managed in laboratory conditions and has an ornamental appeal may assist studies in its reproduction to both supply the aquarium market and help the species conservation in nature. Moreover, these data enable the use of M. oligolepis as a model species in biotechnological applications, such as the germ cell transplantation approach.

Ansiedade, afeto negativo e estresse de docentes em atividade remota durante a pandemia da Covid-19 / Anxiety, negative affect, and stress of faculty in remote work during the COVID-19 pandemic / Ansiedad, afecto negativo y estrés de docentes en actividad remota durante la pandemia de Covid-19

Resumo O trabalho pedagógico remoto emergencial foi implementado em muitas escolas do Brasil durante a pandemia da Covid-19, como forma de diminuir os contatos entre indivíduos e, consequentemente, diminuir a taxa de transmissão da doença, mantendo aulas e atividades didáticas. Em muitas situações, professoras e professores realizaram atividades que descaracterizam sua identidade docente e conflitam com atividades domésticas, produzindo mal-estar e sofrimento. Em pesquisa de coorte em duas fases, o sofrimento psicológico de professores e professoras da Educação Básica foram analisados em função da quantidade de trabalho remoto, do gênero, da quantidade de trabalho doméstico e da experiência prévia. O trabalho remoto emergencial produziu efeitos na Ansiedade-estado, Afeto negativo e Estresse percebido − e essas respostas foram moderadas pela experiência prévia. Mulheres apresentaram maiores respostas que homens, um efeito mediado principalmente pela quantidade de trabalho doméstico realizada pela professora. Os participantes referenciaram principalmente dimensões laborais negativas e dimensões afetivas quando levados a pensar sobre o trabalho remoto, com menor saturação de respostas relacionadas a dimensões laborais positivas, à aprendizagem e a questões político-econômicas. Esses resultados sugerem que o trabalho remoto emergencial exacerba o estresse docente, apontando para precarização do trabalho docente e necessidade de implementação de políticas que mitiguem esses impactos.

Abstract Emergency remote pedagogical work was implemented in many schools in Brazil during the COVID-19 pandemic, as a way to reduce contacts between individuals and, consequently, reduce the rate of disease transmission, maintaining classes and educational activities. In many situations, teachers and teachers carried out activities that mischaracterize their teaching identity and conflict with domestic activities, producing discomfort and suffering. In a two-phase cohort study, the psychological suffering of Basic Education teachers were analyzed as a function of the amount of remote work, gender, the amount of housework and previous experience. Emergency remote work had effects on State Anxiety, Negative Affect and Perceived Stress − and these responses were moderated by previous experience. Women had higher responses than men, an effect mediated mainly by the amount of housework performed by the teacher. Participants mainly referred to negative work dimensions and affective dimensions when led to think about remote work, with less saturation of answers related to positive work dimensions, learning and political-economic issues. These results suggest that emergency remote work exacerbates teacher stress, pointing to the precariousness of teaching work and the need to implement policies that mitigate these impacts.

Resumen El trabajo pedagógico remoto de emergencia se implementó en muchas escuelas de Brasil durante la pandemia Covid-19, como una forma de reducir los contactos entre las personas y, en consecuencia, disminuir la tasa de transmisión de la enfermedad, manteniendo las clases y actividades educativas. En muchas situaciones, profesoras y profesores realizaron actividades que distorsionan su identidad docente y entran en conflicto con las actividades domésticas, produciendo malestar y sufrimiento. En un estudio de cohorte de dos fases, se analizó el sufrimiento psicológico de los docentes de Educación Básica en función de la cantidad de trabajo remoto, de género, de la cantidad de tareas domésticas y de la experiencia previa. El trabajo remoto de emergencia tuvo efectos sobre la Ansiedad-estado, el Afecto negativo y el Estrés percibido - y estas respuestas fueron moderadas por la experiencia previa. Las mujeres tuvieron respuestas más altas que los hombres, efecto mediado principalmente por la cantidad de tareas domésticas realizadas por las profesoras. Los participantes se refirieron principalmente a las dimensiones negativas del trabajo y las dimensiones afectivas cuando se les indujo a pensar en el trabajo remoto, con una menor saturación de respuestas relacionadas con las dimensiones positivas del trabajo, el aprendizaje y las cuestiones político-económicas. Estos resultados sugieren que el trabajo remoto de emergencia agrava el estrés docente, apuntando a la precariedad del trabajo docente y la necesidad de implementar políticas que mitiguen estos impactos.

Social Stress Increases Anxiety-Like Behavior Equally in Male and Female Zebrafish.

Zebrafish anxiety-like behavior was assessed in the novel tank test after the formation of dominant-subordinate hierarchies. Ten pairs of animals were subjected to dyadic interactions for 5 days, and compared with control animals. After this period, a clear dominance hierarchy was established across all dyads, irrespective of sex. Social status affected parameters of anxiety-like behavior in the novel tank test, with subordinate males and females displaying more bottom-dwelling, absolute turn angle, and freezing than dominant animals and controls. The results suggest that subordinate male and female zebrafish show higher anxiety-like behavior, which together with previous literature suggests that subordination stress is conserved across vertebrates.

Chrysin, but not flavone backbone, decreases anxiety-like behavior in animal screens.

Chrysin (5,7-dihydroxyflavone), a nutraceutical flavonoid present in diverse plants, has a backbone structure shared with the flavone backbone, with additional hydroxyl groups that confers its antioxidant properties and effects at the GABAA receptor complex. However, whether these effects are due to the hydroxyl groups is unknown. Here we report the effects of chrysin or the flavone backbone (1 mg/kg) in rats subjected to the elevated plus-maze and the locomotor activity test, as well as in the zebrafish evaluated in light/dark model. Chrysin, but not flavone, increased entries and time in the open arms of the elevated plus-maze, as well as time on white compartment of the light/dark model in zebrafish. These effects were comparable to diazepam, and were devoid of motor effects in both tests, as well as in the locomotor activity test. On the other hand, flavone decreased risk assessment in the light/dark test but increased rearing in the locomotor activity test in rats, suggesting effects threat information gathering; important species differences suggest new avenues of research. It is suggested that the specific effects of chrysin in relation to flavone include more of a mechanism of action in which in addition to its action at the GABAA/benzodiazepine receptor complex also could be involved its free radical scavenging abilities, which require specific research. Preprint: https://doi.org/10.1101/575514; Data and scripts:https://github.com/lanec-unifesspa/chrysin.

Seminal characterization of the Amazonian fire-eye tetra Moenkhausia oligolepis (Günther, 1864).

The seminal characteristics of Moenkhausia oligolepis are described. Three males were induced with a single dose of carp pituitary. Semen was collected 6 h after induction, and diluted in dibasic sodium phosphate extender solution. For motility analysis, 1 µl of diluted semen was added to 10 µl of distilled water to achieve gamete activation. The average duration of total motility was 76.67 s; while the average sperm motility rate at intervals of 15 s was 95.3, 85.3, 59.6, 31.7, 13.0, 4.6 and 1.2%. To determine sperm concentration in samples, 0.5 µl of semen was diluted with 500 µl of glutaraldehyde. An aliquot of 10 µl of this dilution was utilized for cell counting. An average count of 4.97 × 109 ± 3.46 sperm/ml was obtained. Morphological analyses were performed using eosin-nigrosine dye; 20.33% of the sperm were observed to be dead. Live sperm, comprising the other 79.67%, had an average length of approximately 30 µm, with a head diameter of 4.488 ± 0.7 µm; and a flagella plus mid-piece length of 26.071 ± 12.4 µm. Of those sperm, 69% had a normal morphology, while 31% had primary and secondary abnormalities. The observed abnormality rate did not have a detrimental effect on artificial fertilization potential for the species. The description of the seminal characteristics of a species is one of the most important sets of information required for artificial reproduction of fish in captivity. It also contributes significantly to the total biological knowledge of the studied species.

Dopamine and serotonin mediate the impact of stress on cleaner fish cooperative behavior.

Stress is known to modulate behavioral responses and rapid decision-making processes, especially under challenging contexts which often occur in social and cooperative interactions. Here, we evaluated the effects of acute stress on cooperative behavior of the Indo-Pacific cleaner wrasse (Labroides dimidiatus) and the implications of pre-treatment with monoaminergic compounds: the selective serotonin reuptake inhibitor - fluoxetine, the 5-HT1A receptor antagonist - WAY-100,635, the D1 receptor agonist - SKF-38393, and the D1 receptor antagonist - SCH-23390. We demonstrated that stress decreased the predisposal to interact and increased cortisol levels in cleaners, which are alleviated by fluoxetine and the dopaminergic D1 antagonist. Overall, our findings highlight the crucial influence of stress on cooperative behavior.

A model to study orienting responses in zebrafish, and applications towards the emotion-cognition interaction.

Orienting responses (ORs) are whole-organism reflexes that are elicited by innocuous stimuli, and which decrease in magnitude after stimulus repetition. ORs represent relatively simple responses that can be used to study attentional processes, and are modulated by the organism's state, including arousal and activation levels, as well as by emotional processes. Here we describe a simple method to study ORs in zebrafish, a model organism increasingly being used in behavioural neuroscience. After presentation of a static visual stimulus, an OR is elicited, characterized by approaching the stimulus and orienting towards it. After repeated stimulation, OR decreases, suggesting habituation. These responses are qualitatively altered by exposure to a fear-eliciting alarm substance (i.e., derived from the skin of a conspecific), since exposed animals avoid the visual stimulus and orient either away from the stimulus or towards it, but at a distance. The protocol can be used to study orienting responses, as well as the impact of fear and arousal on these reflexes.

NOS-2 participates in the behavioral effects of ethanol withdrawal in zebrafish.

Nitric oxide has been implicated in symptoms of ethanol withdrawal in animal models. Zebrafish have been used as models to study neurobehavioral effects of ethanol (EtOH) withdrawal, but the mechanisms associated with these effects are not yet clear. Adult zebrafish were treated with 1% EtOH for 20 min per day for 8 days, injected with the nitric oxide synthase 2 (NOS-2) inhibitor aminoguanidine (50 mg/kg), and allowed to experience withdrawal (WD) in their hometanks for 7 days. EtOH WD increased anxiety-like behavior in the novel tank test, an effect that was blocked by aminoguanidine. EtOH WD also increased brain levels of nitrite, an effect that was partially blocked by aminoguanidine. These results underline a novel mechanism by which NOS-2 controls anxiety-like responses to ethanol withdrawal, with implications for the mechanistic study of symptoms associated with chronic ethanol abuse. Preprint: https://dx.doi.org/10.20944/preprints201912.0219.v1 Data and scripts: https://github.com/lanec-unifesspa/etoh-withdrawal/tree/master/NOS2.

Involvement of GABAergic system in the antidepressant-like effects of chrysin (5,7-dihydroxyflavone) in ovariectomized rats in the forced swim test: comparison with neurosteroids.

RATIONALE: The absence of ovarian hormones that is characteristic of natural and surgical postmenopause in women is frequently related to such disorders as depression and anxiety. Chronic treatment with the flavonoid chrysin was previously shown to exert antidepressant-like effects in rodents subjected to validate behavioral models. Chrysin has also been shown to have anxiolytic-like properties, but its antidepressant-like effects and mechanism of action in the absence of ovarian hormones remain unknown. OBJECTIVES: To compare the effects of the flavonoid chrysin with the effects of the neurosteroids progesterone and allopregnanolone on depression-like behavior in ovariectomized rats and evaluate the participation of γ-aminobutyric acid-A (GABAA) receptors in these actions. METHODS: Ovariectomized female Wistar rats were subjected to the locomotor activity test and forced swim test. The animals were assigned to eight treatment groups: vehicle, chrysin (1 mg/kg), progesterone (1 mg/kg), allopregnanolone (1 mg/kg), bicuculline (1 mg/kg), and pretreatment with bicuculline followed by chrysin, progesterone or allopregnanolone, respectively. After the treatments, the rats underwent the behavioral tests. RESULTS: Chrysin, progesterone, and allopregnanolone increased the latency to the first immobility and decreased the total immobility time in the forced swim test. The number of crossings and the time spent rearing and grooming decreased from the pretest to test sessions in the locomotor activity test. Chrysin, progesterone, and allopregnanolone only prevented the decreases in rearing and grooming. Bicuculline blocked the effects of chrysin, progesterone, and allopregnanolone in both behavioral tests. CONCLUSIONS: These results show that the GABA-binding site at GABAA receptors participates in the acute antidepressant-like effects of chrysin, similar to neurosteroids, in ovariectomized rats.

Phasic and tonic serotonin modulate alarm reactions and post-exposure behavior in zebrafish.

Current theories on the role of serotonin (5-HT) in vertebrate defensive behavior suggest that this monoamine increases anxiety but decreases fear, by acting at different levels of the neuroaxis. This paradoxical, dual role of 5-HT suggests that a serotonergic tone inhibits fear responses, while an acute increase in 5-HT would produce anxiety-like behavior. However, so far no evidence for a serotonergic tone has been found. Using zebrafish alarm responses, we investigate the participation of phasic and tonic 5-HT levels in fear-like behavior, as well as in behavior after stimulation. Conspecific alarm substance (CAS) increased bottom-dwelling and erratic swimming, and animals transferred to a novel environment after CAS exposure (post-exposure behavior) showed increased bottom-dwelling and freezing. Clonazepam blocked CAS effects during and after exposure. Acute fluoxetine dose-dependently decreased fear-like behavior, but increased post-exposure freezing. Metergoline had no effect on fear-like behavior, but blocked the effects of CAS on post-exposure behavior; similar effects were observed with para-chlorophenylalanine. Finally, CAS was shown to decrease the activity of monoamine oxidase in the zebrafish brain after exposure. These results suggest that phasic and tonic serotonin encode an aversive expectation value, switching behavior toward cautious exploration/risk assessment/anxiety when the aversive stimulus is no longer present.