Advances in Behavioral Testing for Schizophrenia Research Using Animal Models

Abstract

Schizophrenia is a severe and chronic psychiatric disorder affecting more than 21 million people worldwide, significantly impacting cognition, perception, behavior, and emotional regulation. The disorder has a complex etiology involving both genetic predisposition and environmental influences such as prenatal stress, infections, and neurodevelopmental disturbances. Clinically, schizophrenia is characterized by three major symptom domains: positive symptoms, including hallucinations and delusions; negative symptoms, such as anhedonia, avolition, social withdrawal, and reduced emotional expression; and cognitive deficits, including impairments in attention, working memory, and executive function. Understanding the neurobiological basis of these heterogeneous symptoms in humans is challenging; therefore, animal models have become a crucial tool in schizophrenia research. Various behavioral paradigms in rodents are used to mimic specific symptom domains, allowing researchers to investigate underlying mechanisms and evaluate potential therapeutic interventions. These models include genetic manipulations, pharmacological induction using agents such as ketamine and phencyclidine, and developmental models involving prenatal exposure to environmental risk factors like lipopolysaccharide.Behavioral phenotyping in animal models enables the assessment of schizophrenia-related traits, such as sensorimotor gating deficits, social interaction abnormalities, and cognitive impairments. Tests like the prepulse inhibition paradigm, tail suspension test, forced swim test, and maze-based cognitive tasks are widely used to correlate animal behavior with human symptom domains. Although no single model fully replicates the complexity of schizophrenia, combining different approaches provides valuable insights into its pathophysiology, animal models play a critical role in bridging the gap between clinical observations and underlying neurobiological mechanisms, the development of..more effective and targeted treatments for schizophrenia

Keywords

Introduction

Schizophrenia is a chronic and severe psychiatric disorder affecting over 21 million individuals globally, with an estimated lifetime prevalence of approximately 0.4%. It is widely recognized as a multifactorial neuropsychiatric condition, arising from a complex interaction between genetic susceptibility and environmental influences. The disorder most commonly manifests during late adolescence or early adulthood, typically between the late teenage years and the early thirties. Clinically, schizophrenia is characterized by three principal domains of symptoms: positive, negative, and cognitive. Positive symptoms reflect an excess or distortion of normal mental functions and include hallucinations, which involve perceiving stimuli that are not present, as well as delusions, defined as firmly held false beliefs. Other features such as disorganized thought patterns and abnormal perceptual experiences also fall within this category. In contrast, negative symptoms represent a reduction or loss of normal psychological functions. These include diminished emotional expression, decreased motivation, limited speech output, social disengagement, and impaired interpersonal interactions, all of which contribute to significant functional deficits.

Cognitive symptoms encompass impairments in higher-order mental processes, including deficits in attention, working memory, and executive functioning, which substantially affect an individual’s ability to perform daily tasks and maintain independence.

Current pharmacological treatments, particularly antipsychotic medications, primarily target positive symptoms and demonstrate limited effectiveness in addressing negative and cognitive impairments. Furthermore, their overall efficacy is often suboptimal, and their use is frequently associated with considerable adverse effects. Despite extensive investigation, the precise causes and underlying mechanisms of schizophrenia remain incompletely understood. At present, there are no validated biological markers available for definitive diagnosis, and clinical identification relies largely on the assessment of characteristic symptom patterns. Similarly, treatment selection, evaluation of therapeutic response, prognosis, and assessment of functional outcomes are predominantly guided by observable clinical features. [1-6]

1. Open Field Test (Positive Symptoms)

Locomotor activity refers to the movement of an organism within a specific environment. It is commonly evaluated using the open field test, where subjects are placed in an enclosed arena and their activity is assessed by measuring the total distance travelled and the time spent moving throughout the experimental period. This parameter can be easily quantified using automated photocell-based systems or through direct observational methods. More advanced techniques involve assessing a broad range of natural behaviors along with qualitative evaluation of behavioral patterns and repetitive or perseverative actions. In mouse models, locomotor activity is typically measured by placing the animal in a rectangular acrylic chamber with transparent walls, which is entirely unfamiliar to it. The novelty of the environment promotes exploratory behavior, allowing the animal to move freely for a predetermined duration while its movements are recorded either manually or with automated tracking systems.

In multiple validated animal models of schizophrenia, heightened locomotor activity is commonly observed, either under resting (baseline) conditions or when animals are exposed to unfamiliar environments. Developmental models, including maternal immune activation triggered by polyinosinic–polycytidylic acid during gestation, as well as early-life administration of levodopa, have been reported to induce pronounced hyperactivity in the offspring. In addition, a wide range of genetic models demonstrate alterations in movement patterns depending on the specific gene targeted. Many of these models show increased locomotor responses under various experimental conditions, particularly those involving dysfunction in key neurotransmitter systems such as dopaminergic, glutamatergic, and GABAergic pathways. Specifically, alterations in receptor systems play a crucial role in these behavioral changes. For instance, dysfunction of dopamine receptors (especially D1 and D2 receptors) and dopamine transporter (DAT) leads to dysregulated dopamine signaling, contributing to hyperlocomotion. Similarly, disruption of glutamate signaling, particularly through N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptor 5 (mGluR5), is strongly associated with increased locomotor activity and schizophrenia-like symptoms. Other genetic modifications involving proteins such as G protein-coupled receptor 88 (GPR88), dystrobrevin-binding protein 1 (Dtnbp1), collapsin response mediator protein 2 (CRMP2), sterol regulatory element-binding protein 1c (SREBP1c), and glial glutamate/aspartate transporter (GLAST) further contribute to abnormalities in synaptic transmission and neuronal communication, ultimately leading to enhanced activity levels.

However, not all genetic modifications produce changes in locomotor behavior. Certain models, including those with reduced expression of solute carrier family 1 member 1 (SLC1A1), growth arrest-specific protein 7 (GAS7), type III neuregulin-1 (NRG1), and selective phospholipase C-β1 (PLC-β1) knockdown in the medial prefrontal cortex, do not exhibit significant alterations in movement. Despite this, these models may still present other schizophrenia-relevant behavioral deficits, such as impaired sensorimotor gating—often associated with disrupted dopaminergic and glutamatergic receptor activity—or abnormalities in social interaction. Pharmacological models also significantly contribute to understanding locomotor alterations. Drugs such as amphetamine enhance dopamine release and stimulate D2 receptor activity, thereby increasing locomotion. Likewise, NMDA receptor antagonists such as MK-801 and phencyclidine (PCP) impair glutamatergic neurotransmission, resulting in hyperlocomotion and other schizophrenia-like behaviors. However, at higher doses, these substances may produce sedative or anesthetic effects, leading to reduced movement. Additionally, compounds such as tetrahydrocannabinol (THC), which interacts with cannabinoid receptors (CB1), and plant-derived substances like Catha edulis, as well as targeted inhibition of GABAergic neurons (e.g., GAD65 neurons in the ventral hippocampus), further demonstrate the involvement of multiple receptor systems in regulating locomotor activity. Overall, consistent findings across numerous studies highlight increased locomotor activity as a key feature of schizophrenia animal models. This hyperactivity reflects underlying disturbances in interconnected neural circuits, particularly involving dopamine, glutamate, GABA, and cannabinoid receptor systems.[7-10]

2. Prepulse Inhibition (PPI) – Paraphrased Version

Prepulse inhibition (PPI) is a well-established measure of sensorimotor gating and is widely used in studies investigating the genetic basis of schizophrenia. It describes the decrease in the startle response when a mild, non-startling stimulus is presented shortly before a stronger, startling stimulus. In practice, PPI is evaluated by recording the response of an animal to a sudden stimulus, both in the presence and absence of a preceding weaker stimulus. A key strength of this method is its applicability across species, including humans and rodents, making it highly valuable for translational research. In addition, PPI testing is straightforward, reproducible, and typically does not require prior training, as most systems are automated. Despite standardized protocols, several variables can influence PPI outcomes, such as environmental conditions, age, sex, timing between stimuli, type of sensory input, treatment dosage, and genetic background of the subjects. Impairments in sensorimotor gating measured through PPI have been associated with factors like developmental stage, stress exposure, and genetic vulnerabilities linked to schizophrenia. Both genetic and pharmacological animal models have been extensively used to study these deficits and to better understand schizophrenia-like behaviors. Recent research has demonstrated reduced PPI in multiple experimental models. For instance, offspring of BALB/c mice exposed to maternal immune activation using poly show impaired gating. Similarly, deficits have been observed across a range of genetically modified models, including those lacking genes such as SREBP-1c, GPR88, Dtnbp1, and mGlu5, as well as in models involving targeted manipulations like Tmem168 expression in the nucleus accumbens of C57BL/6J mice.hemizygous microdeletion, particularly when combined with pharmacological challenges like amphetamine, along with other variants such as chakragati mice.[10-13]

Negative symptoms

Negative symptoms, also referred to as defect symptoms of schizophrenia, involve a decline in normal functioning that results in disability and a reduced quality of life. These symptoms typically include anhedonia (reduced ability to experience pleasure), avolition (lack of motivation and goal-directed behavior), asociality (withdrawal from social interactions), alogia (limited speech), and blunted affect (reduced emotional expression). The biological mechanisms underlying negative symptoms are complex and not yet fully understood. However, recent research suggests that reduced activity of NMDA receptors, particularly decreased activation of specific NMDAR subtypes, plays a significant role in their development. NMDA receptor hypofunction in cortical interneurons may lead to altered activity of GABAergic interneurons in the ventral tegmental area. This increased interneuron activity can suppress dopamine release in the prefrontal cortex by inhibiting the mesocortical dopaminergic pathway, thereby contributing to negative symptoms. Most currently available treatments for schizophrenia act as dopamine antagonists and are primarily effective in treating positive symptoms, with limited effectiveness against negative symptoms. Therefore, the development of reliable animal models and appropriate methods for evaluating negative symptoms is essential for advancing new therapeutic strategies.

Negative symptoms, also referred to as defect symptoms of schizophrenia, involve a decline in normal functioning that results in disability and a reduced quality of life. These symptoms typically include anhedonia (reduced ability to experience pleasure), avolition (lack of motivation and goal-directed behavior), asociality (withdrawal from social interactions), alogia (limited speech), and blunted affect (reduced emotional expression).

The biological mechanisms underlying negative symptoms are complex and not yet fully understood. However, recent research suggests that reduced activity of NMDA receptors, particularly decreased activation of specific NMDAR subtypes, plays a significant role in their development. NMDA receptor hypofunction in cortical interneurons may lead to altered activity of GABAergic interneurons in the ventral tegmental area. This increased interneuron activity can suppress dopamine release in the prefrontal cortex by inhibiting the mesocortical dopaminergic pathway, thereby contributing to negative symptoms.

Most currently available treatments for schizophrenia act as dopamine antagonists and are primarily effective in treating positive symptoms, with limited effectiveness against negative symptoms. Therefore, the development of reliable animal models and appropriate methods for evaluating negative symptoms is essential for advancing new therapeutic strategies.[14-19]

3. Forced Swim Test (FST)

The Forced Swim Test (FST) is one of the most widely used behavioral assays in animal studies for evaluating the effects of potential antidepressant drugs. In this test, animals are placed in a transparent container filled with water, and trained observers record the duration of immobility over several minutes. An animal is considered immobile when it floats passively, making only minimal movements necessary to keep its head above water.

Although primarily used to study depression-like behaviors, the FST has also been applied to assess negative symptoms of schizophrenia, particularly anhedonia and avolition. Over the years, numerous studies have employed this test, identifying various factors associated with increased immobility in animal models of schizophrenia. These include prenatal maternal immune activation, genetic alterations linked to schizophrenia (such as transcription factor abnormalities and other related genes), changes in receptor-associated proteins, impaired glutathione synthesis, decreased serotonin levels, dysregulated monoaminergic signaling, and dysfunction of the prefrontal cortex. Developmental models, such as offspring of lipopolysaccharide (LPS)-treated mice, have shown increased immobility in the FST. Similarly, several genetically modified models—including Engrailed-2 (En2) knockout mice, EP4 receptor-associated protein (EPRAP) knockout mice, neuronal calcium sensor-1 (NCS-1) knockout mice, neurotensin (NT) knockout mice, and heterozygous reeler mice treated with corticosterone—also demonstrate increased immobility. However, not all genetic models produce the same outcome. Some, such as GRIA1 knockout mice, anaplastic lymphoma kinase (ALK) knockout mice, glutamate-cysteine ligase modifier (GCLM) knockout mice, DBA/2J mice, and receptor protein tyrosine phosphatase gamma (PTPRG) knockout mice, exhibit decreased immobility in the FST. Additionally, certain models, like Grin1 mutant mice, show no significant change in immobility. Pharmacological models have also been widely studied. Administration of NMDA receptor antagonists—such as ketamine, MK-801, and phencyclidine (PCP)—has been shown to increase immobility time in animals.

Overall, across the past decade, most studies using the FST in schizophrenia research have relied primarily on genetic models, followed by pharmacological and developmental approaches. While the FST is commonly used to assess negative symptoms and has been extensively validated, its reliability as a measure of depression-like behavior remains debated. Some studies support its validity using antidepressant treatments, whereas others suggest it may instead reflect psychomotor retardation rather than true depressive states.[19-22]

4. The Tail Suspension Test

(TST: Tail Suspension Test), similar to the Forced Swim Test (FST: Forced Swim Test), is widely used to assess depression-like behavior in mice and is also relevant for evaluating negative symptoms of schizophrenia such as anhedonia and avolition. In this test, mice are suspended by their tails using tape in a way that prevents escape or contact with nearby surfaces. Initially, the animals exhibit active struggling, which gradually transitions into periods of immobility where they hang passively without movement. This immobility is considered an indicator of behavioral despair. Previous studies have thoroughly described the methodology of this test and examined the effects of antidepressants and opioids on immobility behavior.

Recent findings from developmental models of schizophrenia show increased immobility time in mice exposed to lipopolysaccharide (LPS: Lipopolysaccharide) during prenatal stages or early development. Genetic models also demonstrate similar increases in immobility, including mice deficient in sterol regulatory element-binding protein 1c (SREBP1c: Sterol Regulatory Element-Binding Protein 1c), neural cell adhesion molecule (NCAM: Neural Cell Adhesion Molecule), membrane-bound catechol-O-methyltransferase (MB-COMT: Membrane-Bound Catechol-O-Methyltransferase), neuronal calcium sensor-1 (NCS-1: Neuronal Calcium Sensor-1), and neurotensin (NT: Neurotensin). In contrast, some models such as glutamate ionotropic receptor AMPA type subunit 1 knockout mice (GRIA1 KO: Glutamate Ionotropic Receptor AMPA Type Subunit 1 Knockout) and DBA/2J (Dilute Brown Agouti/2 Jackson Laboratory strain) mice exhibit reduced immobility, while others like glutamate ionotropic receptor NMDA type subunit 1 mutant mice (Grin1: Glutamate Ionotropic Receptor NMDA Type Subunit 1) show no significant change. Pharmacological studies further indicate that subchronic ketamine treatment increases immobility, whereas chronic ketamine or phencyclidine (PCP: Phencyclidine) administration does not significantly alter this behavior, although PCP may impair cognitive functions such as working memory. Overall, increased immobility is commonly observed across schizophrenia-related animal models, with genetic factors playing the most prominent role, followed by developmental and pharmacological influences.[22-26]

5. Sucrose Preference Test

The sucrose preference test is a reward-based behavioral assay used to evaluate anhedonia—the inability to experience pleasure—as well as depression- and anxiety-like states. Under normal conditions, rodents naturally prefer sweet solutions, such as sucrose, over plain water when given a choice. However, when exposed to stress or depressive-like conditions, this preference is reduced or lost. In this test, rodents are provided with two drinking bottles: one containing plain water and the other containing a sucrose solution. Their consumption of each is measured over time, and sucrose preference is calculated as the proportion of sucrose intake relative to the total fluid consumed. Studies using animal models of schizophrenia have reported mixed outcomes in this test. A reduction in sucrose preference—indicating anhedonia—has been observed in several models, including mice exposed to prenatal immune activation using poly(I:C), juvenile males treated with levodopa during the perinatal period, cyclin-D2 knockout mice, G72/G30 transgenic mice, and parvalbumin interneuron-specific NMDAR1 knockout mice treated with MK-801. In contrast, some models, such as kainate receptor subunit 4 knockout mice, have shown an increased preference for sucrose. Meanwhile, no significant changes in sucrose preference have been reported in certain models, including Abelson helper integration site 1 heterozygous knockout mice exposed to chronic stress, GluA1 knockout mice, and NRG1 heterozygous knockout mice subjected to repeated psychosocial stress. From a pharmacological perspective, decreased sucrose preference has also been observed in adolescent mice chronically treated with JNJ-28871063, a pan-ErbB kinase inhibitor. Overall, many recent studies suggest a general trend toward reduced sucrose preference in schizophrenia-related animal models. Factors contributing to this decrease include elevated dopamine levels, prenatal immune activation, dysfunction of hippocampal parvalbumin interneurons, alterations in schizophrenia-associated genes, impaired NMDA receptor function in parvalbumin-positive interneurons, and inhibition of ErbB signaling pathways. On the other hand, increased sucrose preference has been reported in studies involving novel antidepressant treatments and models with altered glutamatergic signaling. Interestingly, some schizophrenia models show no changes in sucrose preference, particularly those involving specific gene mutations, glutamatergic dysfunction, or gene–environment interaction paradigms.[27-32]

6. Working Memory and Its Assessment in Animal Models

Working memory is a crucial cognitive function that enables the rapid formation and temporary storage of information from recent experiences, allowing individuals to distinguish new, relevant information from previously stored, outdated data. This function relies heavily on the proper functioning of the prefrontal cortex and plays a key role in reasoning and decision-making. In schizophrenia, working memory is frequently impaired and can be evaluated using clinical cognitive tasks.nIn preclinical research, working memory is assessed in rodents using both spatial and non-spatial tasks. Spatial tasks include the radial arm maze (RAM), T-maze and Y-maze alternation tasks, Morris water maze (MWM), radial arm water maze, Barnes maze, and spatial span tasks. Non-spatial tasks include delayed match-to-sample, delayed non-match-to-sample, delayed stimulus discrimination, and odor span tasks. Poor performance in these tasks may reflect not only working memory deficits but also reduced behavioral flexibility or increased perseverative behavior, both of which are observed in schizophrenia.

Radial Arm Maze (RAM)

The radial arm maze is a widely used tool for assessing spatial working memory across species, including rodents and humans. It typically consists of an eight-armed structure extending from a central platform, with each arm containing a reward. The subject is expected to visit each arm without repetition. Working memory performance is evaluated based on the number of correct entries before revisiting an already explored arm. Most recent studies using the RAM have demonstrated cognitive impairments in various schizophrenia-related animal models, including alpha-CaMKII heterozygous mice, Grin1 mutant mice, heterozygous reeler mice, Homer1 knockout mice, and YWHAE heterozygous knockout mice. However, some models—such as Nrg1 mutant mice, alpha7-nicotinic acetylcholine receptor knockout mice, and mice treated with chronic PCP—did not show deficits in this task. Overall, impairments observed in RAM performance are often linked to genetic factors affecting synaptic plasticity and cognitive function, although not all schizophrenia-associated manipulations produce deficits.

 Y-Maze

The Y-maze is commonly used to evaluate both locomotor activity and spatial working memory, based on the natural tendency of rodents to explore new environments by alternating between maze arms. In this test, the animal is allowed to move freely among three arms, and an alternation is defined as consecutive entries into all three arms without repetition.

Studies have shown that several schizophrenia animal models exhibit reduced spontaneous alternation, indicating impaired working memory. These include mGlu5 knockout mice, mice with inhibited GAD65 neurons, heterozygous reelin-deficient mice treated with corticosterone, and mice subjected to subchronic PCP administration. Genetic risk factors are considered a major contributor to these observed deficits.

 Morris Water Maze (MWM)

The Morris water maze is a widely used task for assessing spatial learning, memory, and working memory in rodents. In this test, animals are placed in a circular pool filled with opaque water and must locate a hidden platform to escape. Training occurs over multiple days, followed by a probe trial without the platform to assess memory retention. A reversal version of the task, in which the platform is relocated, is used to evaluate cognitive flexibility and relearning.

Most studies using the MWM have reported impairments in spatial learning and memory in both genetic and pharmacological models of schizophrenia. These include dysbindin-1B mice, mGlu5 knockout mice, Nlgn2 mutant mice, NRG1 heterozygous mice, mice with disrupted intraflagellar transport, and those treated with substances such as ketamine, MK-801, PCP, or plant extracts like *Catha edulis*. However, some models—such as neonatal ventral hippocampal lesions and zinc transporter 3 knockout mice—did not show significant impairments.

Factors contributing to deficits in MWM performance include genetic abnormalities associated with schizophrenia, disrupted glutamatergic signaling, alterations in synaptic proteins, impaired neuronal development, and pharmacological blockade of NMDA receptors. In contrast, certain developmental or genetic manipulations may not significantly affect spatial memory performance in this task.

Summary

Working memory deficits are a core feature of schizophrenia and can be effectively studied using animal models. Behavioral tasks such as the RAM, Y-maze, and MWM provide valuable insights into cognitive impairments, although results may vary depending on genetic, developmental, and pharmacological factors.

Odor Span Task for Non-Spatial Working Memory

The odor span task is a behavioral paradigm used to evaluate the role of the hippocampus in non-spatial working memory. It has been widely applied in both rats and mice. In this task, animals are trained to dig in scented bowls to retrieve a food reward. Initially, a single scented bowl is presented. In subsequent trials, the animal is exposed to multiple bowls—one containing a familiar scent and another with a novel scent. The reward is always associated with the novel odor, encouraging the animal to ignore previously encountered scents.

As the task progresses, the number of bowls increases. For example, after two scents are introduced, a third novel scent is added in the next trial. The animal must remember all previously presented odors and selectively dig only in the bowl containing the new scent. This process can continue with up to 24 different odors. The bowls are arranged randomly in each trial to prevent the use of visual cues.

The key measure in this task is the “span,” defined as the number of odors the animal correctly remembers before making its first error (i.e., digging in a bowl with a familiar scent). Despite its usefulness, relatively few studies have applied the odor span task in rodent models of schizophrenia. Pharmacological and genetic studies have demonstrated impairments in this task. For instance, subchronic administration of ketamine (10 mg/kg or 30 mg/kg daily for five days) significantly reduced odor span performance. Similarly, α7-nicotinic acetylcholine receptor knockout mice showed deficits in olfactory working memory, evidenced by requiring more training sessions to reach performance criteria, reduced span length, and lower completion rates during testing.

Beyond assessing working memory, the odor span task may also provide insights into verbal learning and memory domains. Although rodents do not possess direct equivalents of human verbal abilities, olfactory cues play a critical role in their communication. Therefore, odor-based tasks may represent the closest functional analogue to verbal information processing in rodents. If this conceptual limitation can be addressed, the odor span task could be considered a model for studying verbal learning and memory. Over the past decade, research using this task has incorporated both genetic and pharmacological approaches in animal models of psychiatric disorders.[33-43]

CONCLUSION

Currently, most research on schizophrenia animal models has primarily emphasized the methods used to create and manipulate these models. However, there is still a lack of comprehensive discussion regarding the behavioral tasks widely applied to characterize them. This review addressed that gap by summarizing the key behavioral paradigms used to evaluate the validity of existing animal models of schizophrenia, along with recent findings from studies employing these approaches.

For the assessment of positive symptoms, locomotor activity and prepulse inhibition (PPI) continue to be the most commonly used behavioral measures. In contrast, negative symptoms are frequently evaluated using paradigms such as the forced swim test (FST), tail suspension test (TST), and sucrose preference test. To assess cognitive impairments, recent studies have largely relied on tasks including novel object recognition, the Morris water maze (MWM), radial arm maze (RAM), Y-maze or T-maze, and the odor span task.

Overall, these behavioral tests effectively replicate key dysfunctions observed in individuals with schizophrenia and help bridge the gap between preclinical and clinical research. Importantly, the development and application of such paradigms play a crucial role in advancing animal model research, thereby contributing to a better understanding of the underlying causes and mechanisms of schizophrenia.

ACKNOWLEDGMENT

We sincerely appreciate all who shared their insights and experiences, and we especially thank Mrs. Samhitha J of Visveswarapura College of Pharmaceutical Sciences, Bangalore, for the opportunity to contribute to this work.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest or competing interests related to this publication.

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