The Neurobehavioral Impact of Zinc Chloride Exposure in Zebrafish: Evaluating Cognitive Deficits and Probiotic Modulation

Abstract

Zinc contamination in aquatic environments has become a growing concern due to its potential to bioaccumulate and induce neurotoxic effects in aquatic organisms. As an essential trace element, zinc plays a crucial role in various physiological processes, but excessive exposure can disrupt the gut-brain axis, leading to cognitive and behavioral impairments. Recent studies have suggested that probiotics may offer protective effects against environmental neurotoxins by modulating the gut microbiota and associated neurological functions. The zebrafish (Danio rerio) has emerged as a valuable model organism for studying the biological mechanisms underlying neurotoxicity and potential therapeutic interventions. This study aimed to assess the effects of probiotics on cognitive impairments induced by zinc chloride (ZnCl₂) exposure in zebrafish. Specifically, zebrafish were exposed to ZnCl₂ at concentrations of 0.5 mg/L and 1.0 mg/L for 96 h, followed by a 7-day post-exposure period to probiotics (Bifidobacterium longum, Bifidobacterium animalis lactis, Lactobacillus rhamnosus). ZnCl₂ exposure at these concentrations is already known to induce behavioral and neuromotor deficits resembling Alzheimer's disease-like symptoms in zebrafish models, making it a suitable model for evaluating the neuroprotective potential of probiotics. Behavioral assessments including sociability tests along with short- and long-term memory evaluations were conducted using EthoVision XT 16 software. Memory tests demonstrated that ZnCl₂ exposure impaired cognitive functions, while probiotic treatment did not significantly ameliorate these deficits. In the social behavior test, ZnCl₂ at 0.5 mg/L resulted in a marked decrease in sociability, whereas exposure to 1.0 mg/L did not induce significant changes. However, post-exposure probiotic administration following ZnCl₂ intoxication at 1.0 mg/L exhibited an anxiolytic effect on zebrafish. These findings suggest that probiotics may exhibit partial neurobehavioral benefits following zinc chloride-induced toxicity, particularly in mitigating anxiety-like behaviors rather than cognitive deficits. Further investigations are needed to elucidate the precise mechanisms by which probiotics interact with the gut-brain axis in the context of heavy metal neurotoxicity.

Keywords: behavior; probiotics; toxicity; zebrafish; zinc chloride.

Figure 1

The visual representation of the experimental design: (A) described experimental design, timeline, and groups. (CTR, control; ZnCl₂, zinc chloride; P, probiotics; h, hours; d, days); (B) the initial experimental setup representation for social preference test; (C) the experimental setup representation for short-term memory test.

Figure 2

Graphical representation of behavioral trends observed in response to zinc chloride and probiotic interventions during the social preference test. (A) Distance moved; (B) Velocity; (C) Inactivity time; (D) Number of entries in the conspecific arm (left arm); (E) Time spent in the maze arms. Data are presented as mean ± SEM, with statistical significance set at p < 0.05 (*).

Figure 3

Graphical representation of behavioral patterns in response to zinc chloride and probiotic treatments during the short-term memory test: velocity (A); distance moved (B); presence in the maze arms (C); baseline characteristics of zebrafish swimming behavior within the treatments (D). PT = Pretreatment. Data are expressed as mean ± SEM and a significance level of p < 0.05 was considered statistically significant (*) and p < 0.01 = **.

Figure 4

Graphical representation of behavioral patterns in response to zinc chloride and probiotic treatments during the long-term memory test. (A) Distance moved; (B) Velocity; (C) Time spent in the sides of the tank (object side vs. novel side). Data are presented as mean ± SEM, with significance levels denoted as follows: *, p < 0.05; ##, p = 0.002; ###, p < 0.001.

Figure 5

Heatmaps visually representing the time spent and activity patterns near the familiar object in the learning period (the silver cube-shaped object) (A) and the new object in the testing period (the black sphere) (B). CTR, the control group; P, the probiotic group; 0.5 mg/L ZnCl₂ and 1 mg/L ZnCl₂, zinc-treated groups; 0.5 mg/L ZnCl₂ + P and 1 mg/L ZnCl₂ + P, probiotic treatment after initial zinc chloride administration. (a—lateral view and b—top view). Where no color variation is present in the tank, the presence of fish was not detected in those locations.