Animal Models of Schizophrenia with a Focus on Models Targeting NMDA Receptors

Schizophrenia is a serious and often devastating disease, affecting approximately 1% of worldwide population. Animal models represent a suitable way for investigating serious brain diseases in preclinical research. Animal models of schizophrenia can be generally divided into several categories that include pharmacological, genetic and neurodevelopmental models. Pharmacological models are usually based on application of specific receptor ligands for neurotransmitters. Genetic models are created by genetic manipulations (mutations; often in laboratory mice). Neurodevelopmental models are induced by manipulations in early stages of life of the laboratory animal. They are manifested later in juvenile or adult age by phenotypes that are similar to schizophrenia symptoms. This review summarizes and discusses pharmacological models of schizophrenia based on application of NMDA receptor antagonists; furthermore, the study focuses on selected genetic and neurodevelopmental models.

tests (Krystal et al., 1994).It is also important to mention that subanesthetic doses of ketamine induce rapid antidepressant effect which persists for a matter of days to weeks (Berman et al., 2000).

Phencyclidine
Phencyclidine is a noncompetitive antagonist of NMDA receptors which has a relatively high affinity for the D2 and 5-HT2 receptors (Kapur and Seeman, 2002).In humans, it induces a syndrome which seemingly resembles schizophrenia and which comprises both its positive and negative symptoms (Javitt and Zukin, 1991).In rodents, it primarily causes stereotyped behavior (Sams-Dodd, 1996), social withdrawal (Corbett et al., 1995), hyperactivity (Kalinichev et al., 2008) and it also disrupts prepulse inhibition (Mansbach and Geyer, 1989) and recognition memory (Egerton et al., 2005).In monkeys, the acute doses of PCP induce deficits in spatial (Boyce et al., 1991) and working memory (Baron and Wenger, 2001) and also in prepulse inhibition (Lynn and Javitt, 2001).Permanent cognitive deficit induced by sub-chronic PCP doses can be attenuated by atypical antipsychotics such as ziprasidone (a dose of 2.5 mg/kg), onlanzapine (a dose of 1.5 mg/kg) and clozapine (a dose of 5 mg/kg) (Abudl-Monim et al., 2006).The classical antipsychotics such as haloperidol (Abdul-Monim et al., 2003) and chlorpromazine have no effect on the deficit (Abdul-Monim et al., 2006).

Dizocilpine (MK-801)
Dizocilpine (MK-801) that was synthesized in 1982 is a phencyclidine-like substance.MK-801 induces hyperlocomotion and impairs cognitive functions.Unfortunately, there is only a small range of doses that can be applied in order to avoid overdosing.It then leads to neurotoxic effects (Olney et al., 1989).In rodents, subchronic exposure to high doses (≥10 mg/kg) induces cell death and neurodegeneration e.g. in olfactory bulbs, dentate gyrus and entorhinal cortex (so called Olney's lesions) (Bender et al., 2010).Likewise PCP, MK-801 also creates a spectrum of motor dysfunctions such as hyperactivity, stereotypy, cognitive deficit or ataxia (Koek et al., 1988) in various species including pigeons, rhesus monkeys and rodents.
In rats the systemic administration of MK-801 causes deterioration in various learning and memory processes, e.g. in passive place avoidance tasks (Ohno and Watanabe, 1996) or in spatial orientation tasks in radial maze (Pitkänen et al., 1995) and in water maze (Whishaw and Auer, 1989).One of the studies found that after MK-801 administration rats find the hidden platform in Morris water maze more slowly than control animals and that they exhibited increased thigmotaxis (they stay closer to the walls of the maze) (Lukoyanov and Paula-Barbosa, 2000).The results of another experiment showed that even a single dose of MK-801 (4 mg/kg) is capable of inducing changes in spatial learning in rats in Morris water maze.These changes subside within five days (Whishaw and Auer, 1989).Furthermore, it was found that the effects of systemic administration of MK-801 on spatial learning in the water maze are extremely difficult to separate from motor and sensory disorders (Ahlander et al., 1999).The results of another study show that when MK-801 is administered to naïve rats that are unfamiliar with the rules of spatial alternation task on rotating arena the working memory worsens and the efficiency of performance in this task is disrupted.However, in animals that undergo a pre-training before MK-801 administration (a dose of 0.12 mg/kg and 0.15 mg/kg) this deficit does not appear (Zemanova et al., 2013).Results of a different study showed that only the highest doses (0.2 mg/kg) of systemically administered dizocilpine induce significant deficit in active place avoidance task learning, while lower doses of MK-801 (0.1 mg/kg) do not affect performance in this task (Stuchlik et al., 2004).Another study found that even 0.15 mg/kg of MK-801 impairs spatial working memory in rodents which, when combined with other findings, suggests that NMDA receptors play a role rather in long-term storage of spatial information (White and Best, 1998).There is a number of studies that connect learning and memory with MK-801 animal model of schizophrenia.One of the studies tested the effect of MK-801 on behavioral (cognitive) flexibility in active place avoidance task and in Morris water maze tasks (MWM) in rats.It turned out that in this schizophrenic behavior model the cognitive flexibility is impaired and that the active place avoidance task is more sensitive to this deficit (in this task the deficit was present already at the dose of 0.08 mg/kg) (Lobellova et al., 2013).Behavioral flexibility is the ability to adapt to the changes in close environment.In schizophrenia patients, this ability is often reduced which, among other things, proved the results of the patients suffering from schizophrenia in virtual decision-making tasks (Han et al., 2012).Changes in attention, behavioral flexibility and adaptation to the new conditions were also observed in animal models of schizophrenia in different experiments, e.g. after exposure to NMDA receptors in attention task (Amitai and Markou, 2010).
Mice in which an animal model of psychosis using MK-801 was induced replaced the components of behavior such as sniffing or pawing by monotonous locomotion.This fact corresponds to the positive symptoms in schizophrenia (stereotypy) and it is associated with excessive dopaminergic activity in the mesolimbic areas (Nilsson et al., 2001).The authors described the changes as overall behavioral primitivization (Nilsson et al., 2001).There is a difference is sensitivity to MK-801 between the sexes: the female rats are more sensitive to this substance probably due to the fact that MK-801 is metabolized in liver and females have lower efficiency of metabolic system (Andino et al., 1999).For the same reason, the females are more sensitive to PCP which leads to its higher concentration in plasma and brain (Nabeshima et al., 1984).It was proved that female rats show 4 to 10 times stronger behavior induced by MK-801 and up to 25 times greater serum and brain concentration of MK-801 than male rats (Andino et al., 1999).
In terms of molecular changes, one study found that MK-801 modifies the expression of NR1 splice variants and NR2 subunits of NMDA (Rujescu et al., 2006) in a similar manner that was observed in schizophrenia (Gao et al., 2000).The study also noted relatively decreased amount of GABAeric parvalbuminpositive interneurons (Rujescu et al., 2006) which is parallel to the changes that were observed in the brains of patients suffering from schizophrenia (Zhang and Reynolds, 2002).In rats, the chronic exposure to MK-801 leads to increased quantity of intracellular glutamate in hippocampus, while in the extra-hippocampal regions no change in concentration was observed (Genius et al., 2013).
It was found that the administration of 5-HT2A/2C receptor antagonists such as ritanserin and risperidone (which is also the D2 receptor antagonist) blocks the cognitive impairments induced by MK-801, while haloperidol, the D2 receptor antagonist, is not able to sufficiently correct the deficit in active place avoidance task in Carousel Maze (formerly known as Active Alothetic Place Avoidance, AAPA).This deficit is induced by MK-801 administration but it effectively blocks hyperlocomotion (Bubenikova-Valešova et al., 2008).Interestingly, after administration of risperidone and haloperidol in intact rats an impaired performance in AAPA could be observed.However, this performance impairment did not occur after ritanserin administration (Bubenikova-Valešova et al., 2008).It turned out that neuroleptics (the antipsychotic agents used for clinical purposes) antagonize behavior induced by MK-801 (Tiedtke et al., 1990) as well as PCP (Sturgeon et al., 1981).This indicates that the behavior induced by NMDA receptor antagonists can be used as complementary model of psychosis in search of new and more effective antipsychotic in schizophrenia treatment.

Neurodevelopmental animal models
Neurodevelopmental animal models are based on neurodevelopmental hypothesis of the emergence of schizophrenia.This hypothesis supposes that a disruption in prenatal or perinatal period of brain development causes dysfunctions of brain connectivity that are manifested by schizophrenia outbreak in early adulthood.This hypothesis is confirmed by e.g.studies aimed at inhibition of NMDA receptors that show that exposure to MK-801 (Harris et al., 2003) and PCP in late fetal or early postnatal period in rats increases the risk of brain damage (Wang et al., 2001).Chronic blockade of NMDA receptors by both MK-801 (Gorter and Bruin, 1992) and PCP during this period causes cognitive deficit and impairs spatial memory in adulthood (Sircar and Rudy, 1998).The time when the inhibition of NMDA receptors increases the cell damage correlates with maximal expression of these receptors.In humans, this period corresponds to the third semester of pregnancy (Lee and Choi, 1992), in rats to the first two weeks of postnatal life (Colwell et al., 1998).Neurodegenerative changes caused by hypofunction of NMDA receptors may serve as models for pharmacotherapy of schizophrenia research (Olney and Farber, 1995).
To the neurodevelopmental models belong also the malnutrition models that address the issue of prenatal malnutrition on brain development (Llorente et al., 2007), models of viral infections (Takei et al., 1995), stress during pregnancy (Fride and Weinstock, 1988), neonatal brain lesions (Lipska and Weinberger, 1993) and other neuronal damages such as postnatal hypoxia after which changes in gene expression of NMDA receptor subunits and reduced prepulse inhibition were observed (Schmitt et al., 2007).
To create a structural model of psychosis in animals the neonatal hippocampal lesions (Lipska and Weinberger, 1993) or medial prefrontal cortex lesions (Jaskiw et al., 1990) are used.The best described neurodevelopmental animal model of psychosis was designed by Lipska and Weinberger.The model consists in performing neonatal excitotoxic ventral hippocampal lesion (Lipska and Weinberger, 1993;1995).After amphetamine application, this lesion causes changes in behavior such as hyperlokomotion (Lipska and Weinberger, 1993).Experimental manipulations with ventral hippocampal lesions are in rats performed on the seventh postnatal day and they result in both temporary (Lipska et al., 2002b) and permanent (Lipska et al, 1993) inactivation of the ventral hippocampus.As a result of these manipulations, hyperactivity (Lipska et al., 2022b), changes in gene expression (Lillrand et al., 1996), deficits in working memory (Lipska et al., 2002b) and impaired prepulse inhibition (Daenen et al., 2003) can be observed in adult rats.It turned out that even temporary inactivation of ventral hippocampus during critical period can trigger behavioral changes similar to those that can be observed in animals with permanent excitotoxic lesions.Since the neonatal disconnection of ventral hippocampus changes the PFC development and plasticity and creates cellular changes that mimic the symptoms of schizophrenia it can represent a potential new model for studying schizophrenia (Lipska, 2004).

Selected genetic animal models
Genetic animal models of schizophrenia slowly displace pharmacological and neurodevelopmental models.The main advantage of these models is the fact that they have potentially grated construct validity (Harrison et al., 2012).There is a wide variety of genes that are, as expected, involved in the pathophysiology of schizophrenia and that can be explored.It is worth mentioning that a number of genetic manipulations aimed at variety of genes are presented as schizophrenia endophenotypes, i.e. isolatable phenotypic characteristics that are bound to a disease, frequently hereditary and often present in healthy blood relatives of these subjects (e.g.Willi et al., 2010).
One of the genes that is associated with schizophrenia is NRG1 (Neuregulin 1) located at chromosome 8p13 (in humans).30cM region around 8p21.1-22 on the chromosome 8p is considered a locus comprising of one or more genes that are co-responsible for the onset of schizophrenia (Pulver at al., 1995).Neuregulins represent a family of growth and differentiation factors encoded by four different genes (NRG-1 to NRG-4) that are bound to ErbB family of tyrosine kinase transmembrane receptors (Papaleo et al., 2012).NRG1 modulates neuronal precursors and cell migration using radial glial cells (Schmid et al., 2003) and increases neuronal survival (Vaskovsky et al., 2000).One of the studies showed that heterozygous mice for NRG-1 receptor of ErbB4 exhibit similar abnormalities in behavior as patients suffering from schizophrenia -e.g.hyperactivity (Gerlai et al., 2000) and that these changes can be partially reversed by clozapine (Stefansson et al., 2002).Recently, the possibility that NRG1 path may be interconnected with NMDA receptors is being considered.The connecting link between these two systems is the fact that ErbB4 receptor is bound to a postsynaptic scaffold protein PSD-95 (Huang et al., 2000) which interacts with NMDA receptors (Kornau et al., 1995).
In vitro stimulation of NRG1 was able to suppress the currents in prefrontal pyramidal neurons induced by NMDA receptors (Gu et al., 2005).Also, the activation of NMDA receptors in prefrontal cortex was significantly suppressed in patients with schizophrenia that exhibited a greater degree of Erb4 -PSD-95 signaling compared to healthy individuals.It follows that more intensive NRG1 signaling may contribute to the hypofunction of NMDA receptors (Hahn et al., 2006).An experiment investigating the short-and long-term effects of chronic blockade of NMDA receptors in the interaction between prefrontal cortex and hippocampus was carried out.24 hours after the last injection of MK-801 the interactions of ErbB4, PSD-95 and NMDA receptors in PFC were increased.However, 12 days after the last dose these effects were not visible, indicating the reversibility of these changes.These results suggest that NRG-ErbB4 signaling may be modulated by repeated blockage of NMDA receptors and provide further evidence of interconnection of these two signaling pathways (Li et al., 2013).
Another possibility for the research is the genetic modification of the NMDA receptors themselves.The receptors are composed of a number of NR1, NR2A-D, NR3A-B subunits (Dingledine et al., 1999).An example of genetic animal model for NMDA receptor hypofunction is the NMDA receptor hypomorphic mouse that has significantly reduced the NR1 subunit (Mohn et al., 1999).These mice exhibit reduced motor activity, deficits in social interactions (Mohn et al., 1999), decreased metabolic activity in the medial prefrontal cortex and hippocampus (Duncan et al., 2002) and prepulse inhibition deficits of an acoustic startle response (Duncan et al., 2004).It was found that clozapine and haloperidol mitigate prepulse inhibition deficits in NR1 hypomorphic mice (Duncan et al., 2006a) and olanzapine reduces their locomotor hyperactivity (Duncan et al., 2006b).The significance of this model for schizophrenia is being challenged because the evidence for abnormalities in genes that cause expression of the NMDA receptor subunits has not been found yet (Nishiguchi et al., 2000).In addition, these animals showed response neither to PCP nor to MK-801 which is contrary to the conclusions that have been found in schizophrenia patients (Krystal et al., 1994).Furthermore, two lines of mouse animal model with a point mutation of glycine binding site on NR1 subunit were created: Grin1 (D481N) and Grin1 (K483Q).The second phenotype confirmed the essential role of NMDA receptor activation in neonatal survival (Kew et al., 2000).The Grin 1D481N/K483Q heterozygous mice exhibited NMDA receptor hypofunction, hyperlocomotion, stereotyped behavior and impaired performance in finding visible platform task in Morris water maze (Ballard et al., 2002).Except that, the NR2 subunit (GluRepsilon1) can also be eliminated.In these mice, NMDA receptors are malfunctioning and mice exhibit hyperlocomotion again (Miyamoto et al., 2001).

Conclusion
The aim of this study was to summarize the findings of hypoglutamatergic animal models of schizophrenia-like behavior.The main reason for this was the fact that the neurodevelopmental hypothesis of glutamate NMDA receptors hypofunction has been recently gaining interest.Currently, the non-competitive antagonists of NMDA receptors such as ketamine, phencyclidine and MK-801 are used for schizophrenia-like behavior modeling in animals.These models have a great potential even though they do not reflect all the symptoms of schizophrenia.These non-competitive antagonists bind to a site on the NMDA receptor ion channel; thereby they block and disrupt their proper functions.Chronic blockade of NMDA receptors can also be caused by application of NMDA receptor non-competitive antagonists in late fetal or early postnatal period.This results in poor brain development and onset of schizophrenia-like behavior in adult animals.In animal models, these antagonists induce a broad spectrum of symptoms relevant to schizophrenia such as locomotor hyperactivity, stereotyped behavior, impaired recognition, and ataxia, deficits in spatial and working memory, reduced prepulse inhibition, changes in glutamate release, and also social withdrawal which is induced by low doses of MK-801.Another promising option that allows researches dealing with NMDA receptors responsibility for the onset of schizophrenia is creating genetically modified animals which exhibit similar behavioral and pharmacological changes as patients suffering from schizophrenia.The contribution of these animal models to schizophrenia research is undeniable; however, knowledge about the origin, etiology and neuropathology of the disease still remains scarce, since due to its complexity a single "ideal" and "universal" animal model that would contain all the symptoms cannot be created.Models can however mimic several important aspects and symptoms of schizophrenia.This fact is also important in finding a safer and more effective medication for the disease.