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Sex is an important variable in translational biomedical research. While overt sex differences have been reported for pain and fear-like behaviors in humans and rodents, these differences in other popular model organisms, such as zebrafish, remain poorly understood. Here, we evaluate potential sex differences in zebrafish behavioral responses to pain (intraperitoneal administration of 5% acetic acid) and fear stimuli (exposure to alarm substance). Overall, both male and female zebrafish exposed to pain (acetic acid injection) show lesser distance traveled, fewer top entries and more writhing-like pain-related behavior vs. controls, whereas female fish more robustly (than males) altered some other pain-like behaviors (e.g., increasing freezing episodes and time in top) in this model. In contrast, zebrafish of both sexes responded equally strongly to fear evoked by acute alarm substance exposure. Collectively, these findings emphasize the growing importance of studying sex differences in zebrafish behavioral and pain models.

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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.

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Anxiety disorders appear to involve distinct neurobiological mechanisms and several medications are available against this mental health problem. However, pharmacological therapeutic approaches display undesirable side effects for patients, particularly when long-term therapy is required. Some evidences have suggested that Coriandrum sativum extract (CSE) provide sedative and anxiolytic effects. We investigate if CSE could attenuate anxiety-like behaviors induced by novelty and alarm substance exposures in zebrafish. Adult zebrafish were injected with vehicle, clonazepam, or CSE (25, 50 or 100 mg/kg) and submitted to novel tank test. At the end, saline or alarm substance was added and anxiety-like responses were recorded. Twenty-four hours after, fish were submitted to the light/dark test. Novelty associated with alarm substance exposure decreased distance traveled and total time mobile in novel tank, and CSE (at 50 and 100 mg/kg) prevented these alterations similarly to clonazepam. Alarm substance reduced the time spent in white compartment (p = 0.0193 as compared with vehicle group). Clonazepam and CSE prevented this anxiogenic effect of alarm substance. CSE presents anxiolytic effects against alarm substance-induced locomotor and anxiogenic responses similarly to clonazepam. These data corroborate with the use of this plant in traditional medicine and provides a putative new pharmacological intervention for anxiety disorders.

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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.

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Several fish species trigger defensive behavioral responses when exposed to chemical cues that indicate predation risk. In these situations, physiological adjustments are also necessary to prepare the organism for a defensive response and increase survival odds. Chemical cues may be derived from predator odor or injured conspecifics. However, little is known about the effects of both cues combined. Therefore, our study evaluated the combined effects of predator odor (PO) and conspecific alarm substance (CAS) on the anti-predatory responses of the pearl cichlid (Geophagus brasiliensis). We set 4 experimental conditions 1) PO + CAS; 2) PO + Distilled Water (DW); 3) DW + CAS; and 4) DW + DW (negative control). Fish exposed to DW + CAS had values of ventilation rate and feeding latency significantly higher than when exposed to the other conditions, which yielded similar responses for both variables. Our data show that the combination of PO + CAS interfered with pearl cichlid behavior and physiology since it abolished CAS defensive responses. These results show that CAS plays an important role on the defensive responses of pearl cichlids, while PO does not, given that fish did not respond to PO by itself. Our findings point to different cue-specific strategies in pearl cichlids when dealing with different chemical cues that indicate predation risk.

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Chemical communication of predation risk has evolved multiple times in fish species, with conspecific alarm substance (CAS) being the most well understood mechanism. CAS is released after epithelial damage, usually when prey fish are captured by a predator and elicits neurobehavioural adjustments in conspecifics which increase the probability of avoiding predation. As such, CAS is a partial predator stimulus, eliciting risk assessment-like and avoidance behaviours and disrupting the predation sequence. The present paper reviews the distribution and putative composition of CAS in fish and presents a model for the neural processing of these structures by the olfactory and the brain aversive systems. Applications of CAS in the behavioural neurosciences and neuropharmacology are also presented, exploiting the potential of model fish [e.g., zebrafish Danio rerio, guppies Poecilia reticulata, minnows Phoxinus phoxinus) in neurobehavioural research.

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The chemical detection of predation risk is direct when based on predator odors, or indirect when an injured conspecific or heterospecific signal it. Physiological adjustments may be necessary in parallel to defensive reactions to cope with an imminent risk. Here, we tested the effects of predator odors and conspecific chemical alarm cues in ventilation response (VR) of frillfin goby, Bathygobius soporator, because this response increases oxygen uptake for supporting behavioral tasks. No VR change was detected in response to odors of predators (catfish) that fed on conspecific, heterospecific fish (tilapia), or were deprived of food and to non-predator (tilapia) that fed chow (non-specific odor control) and odor eluent. The goby's VR, however, increased in response to conspecific alarm cues, but not to heterospecific cues or eluent. Clearly, the VR response in fish depends on the nature of the chemical cue. It is in line with 'threat-sensitive hypothesis' as a chemical cue from an injured prey might mean a foraging predator, whilst the mere presence of a predator odor might not. In addition, because VR can increase, decrease or remains unchanged in response to predation risk in other fish species (including other gobies), we reinforces the species-specific chracteristic of VR responses in fish, regarding the results obtained here for frillfin gobies.

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Fluoxetine is a selective serotonin reuptake inhibitor that increases serotonin concentration in the central nervous system and modulates various systems, including the control of sympathetic outflow and the hypothalamus-pituitary-adrenal. However, it is not yet established whether fluoxetine can modulate the responses to stressors stimulants (physical or chemical) that trigger cortisol response in zebrafish. We demonstrate that fluoxetine blunts the response to physical stress, but not to chemical stress.

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The zebrafish (Danio rerio) is an emergent model organism for assessing fear and anxiety-like phenotypes. The short fin wild type (WT), and leopard (leo) are two zebrafish populations that present several behavioral differences, in which leo displays pronounced defensive responses. Mounting evidence suggests a modulatory role for cholinergic and purinergic signaling in fear and anxiety, but the involvement of these neurotransmitter systems in the behavioral profile of zebrafish is obscure. Here we tested whether the acute exposure to conspecific alarm substance (AS), an experimental protocol that induces fear, alters shoaling behavior, diving response, acetylcholinesterase (AChE) activity, and nucleotide hydrolysis in brain tissue of WT and leo. When four fish were concomitantly exposed to AS extracted from a donor fish of similar phenotype, both populations presented a significant increase of erratic movements without changes in freezing bouts. An increased shoal cohesion and a decreased vertical distribution were observed only in WT exposed to AS. The respective population also revealed a significant increase in AChE and ecto-5'-nucleotidase activities after the exposure period. The comparison of basal endpoints between populations showed that leo displays a higher social cohesion, few vertical transitions and enhanced AChE and ecto-5'-nucleotidase activities. In conclusion, we suggest that the effects of AS on defensive behaviors depend on the population, indicating the existence of distinct neurochemical mechanisms involved. Furthermore, this report shows the first evidence of a potential role of cholinergic and purinergic systems in fear- and anxiety-like responses of zebrafish populations.

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In this study, I show that mianserin, a chemical with serotonin and adrenoceptor antagonist activities, increases fish vulnerability to a potential predator threat, when prey fish must deal with this threat based on conspecific chemical alarm cues. For that, I evaluated whether mianserin, diluted in the water, influences the behavioral responses of Nile tilapia (Oreochromis niloticus) to conspecific skin extract (chemical alarm cues). I found that, while mianserin did not abolished antipredator responses, this drug mitigates some components of this defensive reaction. Thus, a potential decrease in serotonin and adrenergic activities reduces the ability of dealing with predators when perceiving conspecific chemical alarm cues.

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Ammonia has relatively toxic effects on fish and other aquatic organisms. This study examined whether juvenile silver catfish exposed to alarm substances released by conspecifics and predators in water with different ammonia levels modify their behaviour in response to the perceived risk of predation. We used juvenile catfish that were naive to predators. The fish were raised from the larval stage in the laboratory and kept in 40-L aquaria at waterborne NH3 concentrations of 0, 0.05, 0.1, or 0.2 mg L-1 for 10 days. The alarm substances used were predator odour and skin extract from conspecifics. The juveniles were transferred to 2-L aquaria for the antipredator and alarm reaction behavioural tests, which were performed on days 1, 5 and 10 after initial exposure to ammonia. The test aquaria contained a shelter at one end of the tank. The trials consisted of a 10-min prestimulus and a 10-min poststimulus observation period. The results of the study suggest that naive juvenile catfish are able to identify predators and skin extract from conspecifics by odour. In addition, waterborne NH3 levels modify the antipredator response of this species.

Amônia possui efeito relativamente tóxico em peixes e outros organismos aquáticos. Este estudo examinou se juvenis de jundiás modificam seu comportamento em resposta a percepção do risco de predação quando expostos a substâncias de alarme liberadas por conspecíficos e predadores em água com diferentes concentrações de amônia. Foram utilizados juvenis de jundiá nunca expostos a predadores. Os peixes foram criados em laboratório desde a fase larval e mantidos em aquários de 40-L contendo diferentes concentrações de NH3: 0; 0,05; 0,1 e 0,2 mg L-1 por 10 dias. As substâncias de alarme utilizadas foram odor de predador e extrato de pele de conspecíficos. Os juvenis foram transferidos para aquários de 2-L para realização dos testes para comportamento antipredador e resposta à substância de alarme, os quais foram realizados nos dias 1, 5 e 10 a partir do início da exposição à amônia. O aquário teste continha um esconderijo em uma de suas extremidades. Foram feitas observações comportamentais 10-min pré-estímulo e 10-min pós-estímulo. Os resultados do estudo sugerem que juvenis de jundiá nunca expostos a predadores são capazes de identificar predadores e extrato de pele de conspecíficos pelo odor. Além disso, os níveis de NH3 na água alteram a resposta anti-predatória desta espécie.

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Ammonia has relatively toxic effects on fish and other aquatic organisms. This study examined whether juvenile silver catfish exposed to alarm substances released by conspecifics and predators in water with different ammonia levels modify their behaviour in response to the perceived risk of predation. We used juvenile catfish that were naive to predators. The fish were raised from the larval stage in the laboratory and kept in 40-L aquaria at waterborne NH3 concentrations of 0, 0.05, 0.1, or 0.2 mg L-1 for 10 days. The alarm substances used were predator odour and skin extract from conspecifics. The juveniles were transferred to 2-L aquaria for the antipredator and alarm reaction behavioural tests, which were performed on days 1, 5 and 10 after initial exposure to ammonia. The test aquaria contained a shelter at one end of the tank. The trials consisted of a 10-min prestimulus and a 10-min poststimulus observation period. The results of the study suggest that naive juvenile catfish are able to identify predators and skin extract from conspecifics by odour. In addition, waterborne NH3 levels modify the antipredator response of this species.(AU)

Amônia possui efeito relativamente tóxico em peixes e outros organismos aquáticos. Este estudo examinou se juvenis de jundiás modificam seu comportamento em resposta a percepção do risco de predação quando expostos a substâncias de alarme liberadas por conspecíficos e predadores em água com diferentes concentrações de amônia. Foram utilizados juvenis de jundiá nunca expostos a predadores. Os peixes foram criados em laboratório desde a fase larval e mantidos em aquários de 40-L contendo diferentes concentrações de NH3: 0; 0,05; 0,1 e 0,2 mg L-1 por 10 dias. As substâncias de alarme utilizadas foram odor de predador e extrato de pele de conspecíficos. Os juvenis foram transferidos para aquários de 2-L para realização dos testes para comportamento antipredador e resposta à substância de alarme, os quais foram realizados nos dias 1, 5 e 10 a partir do início da exposição à amônia. O aquário teste continha um esconderijo em uma de suas extremidades. Foram feitas observações comportamentais 10-min pré-estímulo e 10-min pós-estímulo. Os resultados do estudo sugerem que juvenis de jundiá nunca expostos a predadores são capazes de identificar predadores e extrato de pele de conspecíficos pelo odor. Além disso, os níveis de NH3 na água alteram a resposta anti-predatória desta espécie.(AU)

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