Comorbidities between gastrointestinal diseases and psychiatric disorders have been widely reported, with the gut-brain axis implicated as a potential biological basis. Thus, dysbiosis may play an important role in the etiology of schizophrenia, which is barely detected. Triple-hit Wisket model rats exhibit various schizophrenia-like behavioral phenotypes. The present study aimed to compare the diversity and abundance of gut microbiota in Wisket model and control rats; furthermore, to correlate the microbial taxonomic profiles to indices of behavioral change. Tail-flick and Ambitus tests were used to assess acute heat pain sensitivity, and record exploration and locomotor activity along with motivation in young adult, control and Wisket model rats. Fecal microbiota composition was profiled by deep sequencing of bacterial 16S rRNA, and it was correlated to behavioral phenotype. Wisket rats exhibited significantly decreased pain sensitivity, lower locomotor activity and exploration, and impaired motivation compared with controls. No significant differences were observed in bacterial alpha diversity between the groups; however, clear differences in community structure were observed. Wisket rats showed decreases in several genera of Firmicutes and Saccharimonas, and increases in Bacteriodetes and Helicobacter phyla compared with controls. Correlation analysis revealed significant associations between the microbiota profile and the behavioral phenotype. This is the first demonstration that fecal microbiota composition is markedly altered in a triple-hit schizophrenia rat model, suggesting the contribution of the microbiota-gut-brain axis in the development of the schizophrenia-like behavioral phenotype. Thus targeting the gut microbiota may be a novel approach to treat such impairments.

The limited efficacy of antipsychotics in treating the negative and cognitive symptoms of schizophrenia has prompted the exploration of adjuvant therapies. Several drugs developed for other indications—including caffeine, metformin, and furosemide—have shown procognitive potential. This study evaluated the effects of these agents on behavioral parameters using the reward-based Ambitus test, and on the cerebral D2 dopamine receptor (D2R) expression and binding. The drugs were administered individually and in combination in a schizophrenia-like triple-hit animal model (Lisket rats), derived from the Long Evans (LE) strain. Lisket rats received 14 days of drug treatment via drinking water; water-drinking LE rats served as the controls. The Ambitus test was conducted before treatment and on days 11–14. Caffeine enhanced activity without affecting learning or memory. Metformin and furosemide reduced exploratory behavior but improved reference memory; these effects were inhibited by caffeine co-administration. Although no statistically significant behavioral differences were found compared to water-treated Lisket rats, a trend toward reduced exploratory visits was observed in the triple-combination group. Lisket rats exhibited moderately reduced D2R binding in the cortex and increased binding in the hippocampus. Caffeine alone and in combination enhanced hippocampal D2R binding, while furosemide increased cortical D2R expression. This study is the first to highlight the behavioral and molecular effects of these non-antipsychotic agents in a schizophrenia model, supporting their potential for adjunctive use.

Modeling schizophrenia in rodents is challenging because of the disorder's complexity and multifactorial etiology. This study aimed to elucidate the transgenerational consolidation of schizophrenia-relevant behavioral alterations in a triple-hit rat model (Lisket) developed on the behaviorally active Long Evans (LE). The Lisket line was established by combining post-weaning social isolation, repeated ketamine exposure, and selective breeding based on behavioral traits across 13 generations. A total of 1154 rats were assessed using repeated tail-flick test to measure pain sensitivity and the reward-based Ambitus test to evaluate locomotor activity, exploratory behavior and reward-guided task performance. Detailed sex-specific and individual-level analyses were performed in the final generation. Over six years, Lisket rats showed a gradual emergence of decreased pain sensitivity, reduced behavioral activity, and altered reward-guided task performance, potentially reflecting a combination of motivational, exploratory, and cognitive influences. Most parameters were significantly associated with generation, and the phenotype was generally more pronounced in males. In the final generation, principal component analysis showed a clear separation between the two lines, consistent with a more consolidated behavioral phenotype in Lisket rats. Altered response dynamics across repeated trials were also observed in the Lisket animals, while individual-level profiling showed that even animals with relatively preserved task performance often displayed deficits in other behavioral domains. These findings suggest that the Lisket line may provide a useful preclinical model for studying neurobiological mechanisms and interventions relevant to schizophrenia-associated behavioral alterations, particularly negative symptom-like and reward-guided performance-related domains.