Animal models
The unique complexity of the human brain in both function and structure poses challenges for schizophrenia research. Studies of the living patient's brain primarily rely on neuroimaging and cognitive testing, while direct analysis of brain tissue is restricted to post-mortem investigations. Factors such as ongoing environmental stress, genetic influences and drug effects in patients complicate research, necessitating the development of reliable animal models. An effective model should encompass three core characteristics: Face validity, construct validity and predictive validity (91). Face validity requires that the model replicates the symptoms of schizophrenia, while construct validity ensures the model mimics the structural and functional abnormalities observed in the disease. Predictive validity ensures that therapeutic interventions can reverse the disease-like phenotype. Animal models, including both invertebrates and vertebrates, such as non-human primates, offer valuable tools for investigating the mechanisms underlying schizophrenia risk. The MATRICS program, developed by the National Institute of Mental Health, identified seven domains relevant to schizophrenia and proposed corresponding rodent-based assays (92).
Established high-risk factors, such as maternal infections and early neurodevelopmental disorders, play a critical role in the pathophysiology of schizophrenia. Animal models addressing these factors often involve perinatal and/or early postnatal environmental manipulation, or the administration of medications, followed by longitudinal studies of offspring development. One of the earliest approaches to modeling schizophrenia in animals involved inducing ventral hippocampal lesions in rats through localized toxin injections on postnatal day 7. Another widely used model is the administration of methylazomethanol (MAM) to pregnant rats (93), which induces neuroanatomical, electrophysiological and behavioral alterations in offspring, with the timing of MAM exposure-particularly on gestational day 17-being a critical determinant of model fidelity (94).
Inducing maternal immune activation (MIA) in pregnant rodents is another widely used strategy for constructing schizophrenia models. Strong evidence suggests that rodents and primates subjected to MIA during gestation produce offspring exhibiting anatomical, neurochemical, electrophysiological and behavioral alterations consistent with schizophrenia (95). Using this model, Steullet et al (96) demonstrated that changes in parasympathetic interneurons and their associated perineuronal networks (extracellular matrix structures critical for structural and synaptic plasticity) in the prefrontal cortex and hippocampal formation were linked to cognitive deficits, findings aligning with autopsy reports from patients with schizophrenia (97).
The most commonly utilized pharmacological agents in animal models of schizophrenia are dopamine enhancers and noncompetitive malondialdehyde (NMDA) receptor antagonists. Amphetamine, a dopamine-enhancing agent known to induce psychosis in humans, does not cause social impairment in rodents with continuous administration, although it does affect certain forebrain-associated cognitive functions (98). Studies have shown that amphetamine impairs cholinergic basal neurons in rat cerebral cortical projections, suggesting that dysregulation of this activity is related to attentional dysfunction (99-101). Wearne et al (102) identified 96 differentially expressed proteins in the prefrontal cortex of methamphetamine-treated rats, with 20% linked to schizophrenia pathogenesis. Additionally, certain dopamine enhancers are known to elevate tumor necrosis factor α (TNFα) and MDA, signaling increased inflammation and oxidative stress (103). NMDA receptor antagonists are often used in rodents to simulate the hypofunction of NMDA glutamate receptors observed in patients with schizophrenia (104). The NMDA receptor antagonist pentachlorophenol (PCP) induces hallucinations, delusions, speech impoverishment and cognitive deficits in humans, while in rodents, PCP causes social withdrawal and cognitive impairments.
Advances in human genome-wide studies have recently revealed a growing list of schizophrenia-associated and candidate genes (105), emphasizing the importance of genetic factors in the development of animal models. The identification of these candidate genes has significantly contributed to refining and enhancing schizophrenia model systems (106,107). Certainly, animal models used in modern research must obtain approval from the Animal Research Ethics Committee and comply with the animal experiment guidelines or directives formulated by relevant institutions.
Cellular models
In the post-genome-wide era of schizophrenia, the development of cellular models incorporating schizophrenia-associated variants has become a crucial tool for understanding the disease. Both autopsy and in vivo neuroimaging studies have highlighted neuronal and glial cell abnormalities as prominent features of schizophrenia.
SH-SY5Y neuroblastoma cells are widely used as an in vitro model for neuropsychiatric research due to their low cost, ease of culture and reproducibility (108). These cells resemble immature neurons, and human-derived SH-SY5Y neuroblastoma cells can differentiate into neuronal cells when exposed to various substances, with both undifferentiated and differentiated SH-SY5Y cells capable of secreting dopamine (109). While some current research questions the maturity of SH-SY5Y-derived neurons, they express well-established neuronal maturation markers, including neuronal nuclei, high-molecular-weight neurofilament, microtubule-associated protein 2 and growth-associated protein 43 (110,111). Furthermore, there is substantial evidence supporting the suitability of SH-SY5Y-derived neurons as functional and morphological neuronal models.
Artificially manipulating SH-SY5Y cells to construct cellular models greatly benefits research into schizophrenia at the cellular and molecular levels. SH-SY5Y cells treated with the NMDA antagonist MK-801 are commonly used to model neuronal damage seen in patients with schizophrenia. MK-801 downregulates the sirtuin 1/microRNA (miRNA)-134 signaling pathway and Ca2+ influx, replicating dysfunction in the prefrontal cortex (112). Additionally, SH-SY5Y cells are utilized to investigate the molecular mechanisms underlying reduced neurite spines, a known susceptibility factor for schizophrenia that is also observed in postmortem brain samples from patients (113). Lentiviral infection of SH-SY5Y cells with short hairpin RNA targeting the schizophrenia susceptibility gene DISC1 results in mitochondrial fragmentation and respiratory defects. Studies on DISC1 in SH-SY5Y cells suggest that DISC1-depleted cells fail to maintain proper mitochondrial oxidative phosphorylation complex function, and mitochondrial abnormalities may have a role in the onset of schizophrenia (114). SHANK2, a gene encoding a protein essential for the formation and development of glutamatergic synapses, has also been implicated in schizophrenia pathogenesis (115). Mutations in SHANK2 in SH-SY5Y cells impair early neuronal differentiation, alter cell growth properties and reduce both pre- and post-synaptic protein expression (116). Collectively, these studies strongly support the use of SH-SY5Y cells as a reliable model for investigating schizophrenia. Additionally, SH-SY5Y cells have proven valuable as a screening platform for potential antipsychotic drugs (117).
Induced pluripotent stem cells (iPSCs) were initially utilized to model diseases linked to highly penetrant genetic variants with significant phenotypic effects, and more recently, iPSC-derived neurons have been employed to construct models of psychiatric disorders. Although iPSCs are not commonly used to model schizophrenia-related processes directly, genetic variants associated with schizophrenia can substantially affect iPSCs. For instance, the 22q11.2 deletion has been shown to reduce the proliferation rate of iPSCs in culture (118). Furthermore, the study of patient-derived iPSCs enables the screening of genetic variants that may not be detectable through human genome-wide studies, while also shedding light on the roles of relevant candidate variants. Although the reproducibility of iPSC models remains a subject of debate, careful selection of iPSC lines for reprogramming, as well as thoughtful consideration of subjects and controls, can mitigate many of their limitations (119).
iPSC-derived neurons mainly include neural progenitor cells (NPCs), glutamatergic neurons, dopaminergic (DAergic) neurons and γ-aminobutyric acid-secreting (GABAergic) neurons. Studies involving NPCs have broadened the understanding of schizophrenia, highlighting its complexity as a neurodevelopmental disorder. The glutamatergic neurons model is based on the glutamatergic hypothesis, where SNP-4bp mutations in DISC1 in glutamatergic neurons result in reduced presynaptic labeling and defective neurotransmitter release. Notably, the mutant phenotype can be reversed by restoring the DISC1 sequence, thereby demonstrating how synaptic defects contribute to schizophrenia (120). The DAergic neurons model is linked to the dopaminergic dysfunction hypothesis, which remains central to schizophrenia research, supported by both animal models and human studies (121). The iPSC-derived DAergic neurons model helps elucidate the molecular mechanisms underlying the relationship between genetic variants and dopaminergic dysfunction in patients with schizophrenia. While most studies on iPSC-derived GABAergic neurons have focused on Huntington's disease, research has indicated the downregulation of key GABA pathway genes in GABAergic neurons derived from patients with schizophrenia, suggesting there is still significant room for further exploration of GABAergic neurons in schizophrenia (122).
Non-neuronal cells can be reprogrammed into neuronal cells or used directly as cell models. It has been demonstrated that fibroblasts can be directly converted into patient-specific inducible neurons, such as glutamatergic and GABAergic neurons, through transcription factor overexpression and the addition of miRNAs (123). Non-neuronal cells, like HEK293 cells, can also serve as models for studying genes and proteins associated with schizophrenia. The primate-specific mutant isoform Kv11.1-3.1 of the brain voltage-gated potassium channel exhibits aberrant folding and impaired migration to the plasma membrane, which correlates with prolonged neuronal action potentials observed in patients with schizophrenia. Researchers have developed a platform for screening drugs that restore Kv11.1-3.1 function using HEK293 cells (124). Studies on the DPYSL2 isoform in HEK293 cells have shown that alterations in risk alleles can lead to changes in cellular morphology, affecting synaptic transmission and correlating with schizophrenia susceptibility (125,126).
In vitro models informed by human genome-wide studies offer significant insights into the brain pathology of schizophrenia and its underlying molecular mechanisms. Additionally, these models provide valuable tools for screening potential novel antipsychotic drugs and validating therapeutic candidates. As schizophrenia models continue to evolve, their role in advancing research and drug development will become more pivotal.