Anxiety disorder represents one the most well characterized behavioral alterations in subjects submitted to acute or chronic stress (Egan et al., 2010; Piato et al., 2011; Etaee et al., 2019; Harada et al., 2024). As described by World Health Organization (2022), post-traumatic disorders, including anxiety disorder, affect millions of people around the world and represent a serious problem of public health in different countries. Traumatic events induce severe pattern anxiety disorder characterized by intense somatic symptoms (e.g., palpitations, dizziness, dyspnea) as well as emotional and cognitive impairments (e.g., negative affect, fear, worry, and rumination; Szuhany and Simon, 2022; Andreescu, 2015). Together, these symptoms lead many people to suicide, social isolation or retirement (Behrens-wittenberg and Wedegaertner, 2021; Nepon et al., 2011; Wolters et al., 2023. In face of these data, many efforts are focused on clarifying the brain alterations related with the development of anxiety disorder triggered by different acute stress.

It is well documented that the behavioral aspects inherent to anxiety are mediated by alterations in the homeostasis of different neurotransmitters system (Sandford et al., 2001; Millan, 2003) as well as in anatomically distinct areas of the brain (Anand and Shekhar, 2003; Robinson et al., 2019). Among them, the role of gamma-aminobutyric acid (GABA) stands out, whose inhibitory action on the limbic system decreases defense reactions against threats (Nutt and Malizia, 2001; Lydiard, 2003; Kalueff and Nutt, 2007; Nuss, 2015). Alterations in GABA levels are believed to affect the susceptibility of the CNS to the actions of glutamate, resulting in hyperactivation of systems associated with the classic symptoms of anxiety, making these neurotransmitters fundamental in the development of the disorder (Kalueff and Nutt, 2007; Wierońska, 2011; Nuss, 2015). Currently, one of the pharmacological strategies for the treatment of anxiety disorders is based on positive modulation of GABAergic receptors to restore control of the excitability of local circuits, as observed with the use of benzodiazepines (Nuss, 2015). Furthermore, due to the involvement of other neurotransmission systems, such as noradrenergic and serotonergic, antidepressants are also frequently prescribed (Fluyau et al., 2022; Lee and Stein, 2023). However, despite their efficacy, these drugs are associated with adverse effects, such as dependence, sedation, memory impairment, and drowsiness (Baldwin et al., 2013; Sinha et al., 2017). Given these limitations, there is a growing need to explore new modulators that can alter neuronal function and behavioral processes with better tolerability. Since benzodiazepines exert their anxiolytic effects through increased GABA-mediated inhibitory transmission, an alternative strategy would be to reduce glutamatergic excitatory neurotransmission (Wierońska, 2011).

Previous reports have already described that different kinds of aversive stimuli can trigger anxiety disorders (Egan et al., 2010; de Abreu et al., 2021; Harada et al., 2024). However, it remains unclear how stressors of different natures (mechanical, physical, or chemical) alter brain neurochemistry, evoking various types of anxiety disorder. In this study, we hypothesize that different aversive stimuli elicit distinct responses in the GABAergic and glutamatergic systems, associated with varying intensities of anxiety-like behavior. To test this hypothesis, we used zebrafish (Danio rerio), a well-established animal model for investigating the neurobiology of various neurological disorders (Panula et al., 2010; Maximino et al., 2011; Stewart et al., 2012; Fontana et al., 2020).

In the present study, we observed for the first time that stress-induced anxiety behavior through different stressors exposure not only results in varying magnitudes of anxiety phenotypes but also elicits diverse neurochemical responses. Those results demonstrate the complexity of anxiety disorders, emphasizing that both behavioral and neurochemical outcomes are highly context dependent.

It is well known that in the Novel Tank Test, vertical locomotion is a parameter used to assess anxiety-like behavior due to the animal’s tendency to escape from the water surface (Kysil et al., 2017). Previous results from our group have already shown that when fish are exposed to Acute Restraint Stress there is a decrease in the time spent on top (Assad et al., 2020; Lucas Luz et al., 2021). Taken together, these studies suggest that increased exploration of the upper zone is related to reduced anxiogenic behavior, and remaining in the lower zone may reflect anxiety-like, depressive-like, or fear-like behavior. Our results align with this understanding, showing that animals exposed to acute stress exhibited a robust anxiogenic effect in the Novel Tank Test, including significantly less time spent at the top. However, despite the overall decrease in time spent at the top across all exposed groups, the animals exposed to the Alarm Substance and the group subjected to Chasing Net showed an even more pronounced reduction in this parameter. Previously, Egan et al. (2010) observed that exposure to the alarm substance acutely, but not chronically, was capable of inducing anxiety-like behavior in a robust manner, being observed by the reduction in time spent at the top, increase in freezing time and erratic movements.

Zebrafish, when faced with aversive conditions tend to use defense strategies that range from preference for safe environments at the bottom to increased freezing time and erratic movements during swimming (Kalueff et al., 2013). Therefore, the data from the present study confirm this understanding since the stressors used could induce an increase in freezing time. However, this effect was more pronounced in the groups exposed to physical or chemical stress stimuli compared to the group exposed to spatial restrictions or not exposed. Mongeau et al. (2003) propose that animals with a low level of anxiety tend to flee when faced with an aversive stimulus, while highly “anxious” animals freeze. Added to this, we also observed that when exposed to the stressors used here, the animals increased the frequency of erratic swimming. And interestingly, we again observed a differential behavioral response between the stressors, where the CN and CAS groups presented higher frequencies compared to the ARS, with high intensity in the CAS group. These factors appear to have been decisive in the impairment of locomotion in animals subjected only to physical and chemical stress, observed by the increase in the average speed covered by the animals. Furthermore, due to the increase in erratic movements, marked by unpredictable and rapid changes in swimming direction, these two groups (CN and CAS) presented a higher maximum speed, unlike animals controlled under stress due to spatial restriction. Together, these findings support the hypothesis that exposure to different stress stimuli induces varying magnitudes of anxiety-like behavior. Based on this, we hypothesize that this observed phenomenon is the result of the involvement of different brain regions and mechanisms that evaluate the nature and intensity of the stimulus to develop the specific behavioral response.

The amygdala represents the limbic structure involved in the generation of anxiety-like behavior induced by acute stress, where each nucleus is specialized in distinct neural connections (Tovote et al., 2015). In zebrafish, the subpallial amygdala is located in the regions of the ventral telencephalon (dorsal nucleus-Vd, supracommissural nucleus-Vs and post-commissural nucleus-Vp) associated with the mammalian central amygdala, possessing a larger population of GABAergic neurons (Ganz et al., 2012; Porter and Mueller, 2020), while the pallial amygdala is located in the dorsal telencephalon (medial dorsum-Dm and lateral dorsum-Dl), related to the mammalian basolateral amygdala, where the majority of neurons are glutamatergic (Wullimann and Mueller, 2004; von Trotha et al., 2014). It is well established that the GABAergic system plays a crucial role in anxiety disorders. Reduced levels of this neurotransmitter in the limbic system are often associated with diminished inhibitory control, which can lead to increased excitability and contribute to the development of the primary symptoms observed in anxiety disorders (Wierońska, 2011). Added to this, we observed a strong positive correlation between the time that animals remained at the top and the release of GABA. This finding suggests that animals that spent more time on top, reflecting less pronounced anxiety-like behavior, presented higher levels of GABA. In contrast, we demonstrated that only physical and chemical stressors were able to increase glutamate release in the zebrafish brain, which was not observed in the group exposed to the stressor due to spatial restriction. Thus, we observed a moderate negative correlation between time on top and glutamate release, where animals that exhibited more pronounced anxiety-like behaviors, such as those in CN and CAS groups, presented higher glutamate levels. These results highlight an inverse relationship between the inhibitory and excitatory systems that plays a crucial role in the regulation of stress responses, directly influencing the exploratory behavior and anxiety levels of the animals. In this sense, our data are linked to those found by Assad et al. (2020), who observed that acute restraint stress can inactivate the Vs subnucleus of the zebrafish telencephalon and consequently reducing GABA levels but does not alter the activation of the Dm and Dl regions, not being able to modulate the glutamate levels. These data suggest that the anxiety-like behavior induced by acute restraint stress was mediated only by GABAergic dysfunction. Thus, our hypothesis is that the low impact of the response to a spatially constrained stressor (ARS) may be related to its exclusive site of activation, while physical (CN) and chemical (CAS) stressors may depend on other pathways for perception of the stimulus.

In this regard, the Dm region receives sensory projections from the thalamus (somatosensory, visual, auditory) and the preglomerular complex (gustatory and odor) and sends projections to the subregions of the ventral nucleus and to the Periaqueductal Gray Matter (PAG; Northcutt, 2006; Lal and Kawakami, 2022). Calcium imaging studies show that the Dm region is activated in response to odors (Diaz-Verdugo et al., 2019; Bartoszek et al., 2021). Therefore, the increase of glutamate in the Dm subnucleus after a stressful stimulus can result in the activation of the PAG, which is specialized in the generation of defensive behavior, associated with more extreme defense reactions. This may be an important pathway in mediating anxiety-like behavior induced by chemical and physical stressors.

Despite physiological differences between zebrafish and humans, zebrafish exhibit anxiety-like behaviors in response to stressors that closely resemble those observed in mammalian and human models (Costa et al., 2023; Maximino, 2010). For instance, zebrafish exposed to physical and chemical stressors show anxiety-like behaviors like those observed in mice, indicating that these responses are governed by shared neurobiological mechanisms (De Abreu et al., 2014; Harada et al., 2024). This is further supported by studies demonstrating that stress-induced behaviors in zebrafish, such as reduced time spent in the upper zone of a tank are analogous to anxiety responses in other animal models (De Abreu et al., 2014; Kalueff et al., 2013). Our study advances this understanding by illustrating those different types of stressors—spatial, physical, or chemical—elicit varying magnitudes of behavioral and neurochemical responses in zebrafish. Specifically, our data show that distinct stressors activate different neurobiological pathways, as evidenced by variations in exploration time and neurotransmitter levels, including GABA and glutamate. These findings reflect the complexity of human anxiety disorders, as described in the DSM-V (American Psychiatric Association, 2013), where a range of stressors and anxiety manifestations are linked to intricate neurobiological mechanisms. The variations in GABA and glutamate levels in response to different stressors align with observations in mammalian models, where neurotransmitter systems are pivotal in regulating anxiety. This suggests that zebrafish can offer valuable insights into the neurochemical foundations of anxiety and support the development of more targeted pharmacological treatments. Understanding how different stressors affect these pathways can propose therapies tailored to the specific nature of the aversive stimulus.

Currently, the most effective anxiolytic medications are based on modulation of the GABAergic system, either by inducing an increase in the bioavailability of GABA or an increase in the affinity of the neurotransmitter to its GABAa receptor, such as benzodiazepines. However, due to the involvement of the glutamatergic system in the generation of anxiolytic behaviors, there is a need to carry out studies that aim to modulate this system with the aim of uncovering the mechanisms inherent to anxiety disorder, as well as improving the pharmacology currently used for treatment.

This study strongly suggests that different aspects of anxiety-like behavior are the result of differential neurochemical changes based on the action of GABA and, mainly, glutamate. That is, both behavioral and neurochemical results are highly context-dependent, supporting our initial hypothesis. Thus, we believe that the action of glutamate in anatomically distinct areas of the brain may be linked to different aspects of the anxiety response. Exploring this issue may open new pharmacological possibilities that consider the nature of the aversive stimulus and offer a deeper understanding of the pathogenesis of anxiety disorders induced by acute stress.