Combined Aluminum Chloride and Lead Acetate Exposure Induces Anxiety-like Behavior, Cognitive Impairment, and Cholinergic–Oxidative Dysregulation in Adult Zebrafish: Optimization of a Dementia-like Model

Dementia-related neurotoxicity induced by environmental metals remains a public health concern, and experimentally tractable vertebrate models that integrate behavioral and molecular endpoints are needed. Here, we established an adult zebrafish (Danio rerio) co-exposure paradigm using aluminum chloride (AlCl3; 0.75 and 1.50 μM) and lead acetate (AcPb; 0.26 and 0.53 μM) and assessed anxiety-like behavior and cognition together with cholinergic and oxidative stress biomarkers. In the Novel Tank Diving Test, co-exposure increased latency to the top zone and reduced vertical exploration (top/bottom time ratio), with enhanced freezing, while average speed and total distance were not significantly altered, indicating an anxiogenic phenotype without major locomotor impairment. In the Novel Approach Test, the high co-exposure condition (AlCl3 1.5 μM + AcPb 0.53 μM) produced the strongest avoidance of the inner zone and increased immobility, effects attenuated by mexazolam (10 µM). Cognitive performance was markedly impaired under combined exposure: in the Novel Object Recognition Test, the novel-object preference index decreased from 64.83% (control) to 35.75–36.73% (co-exposure; p = 0.0016), whereas galantamine improved preference (58.60%). In the Y-maze test, spontaneous alternation dropped from 78.22% (control) to ~42.90–44.94% in AlCl3 200 and both co-exposures (p = 0.0048), and time in the novel arm decreased from 26.09 s to 10.53–11.35 s (p < 0.0001). Biochemically, metals inhibited brain acetylcholinesterase, most strongly under high co-exposure (7.60 vs. 15.92 nmol ATCh/min/mg; ~52%), and induced severe oxidative stress, evidenced by decreased superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione, alongside increased malondialdehyde (MDA) and protein carbonyls (with MDA doubling to 14.90 vs. 7.04 nmol/mg). Pearson correlations supported coupling between the cholinergic/redox axis and novelty-related behaviors. Overall, this easy-to-implement zebrafish Al–Pb model provides a practical platform for mechanistic studies and in vivo screening of candidate neuroprotective agents.