Mice with subtle reduction of NMDA NR1 receptor subunit expression have a selective decrease in mismatch negativity: Implications for schizophrenia prodromal population
Introduction
Recent evidence suggests that several core feature of schizophrenia may result from decreased glutamatergic activity. Many of the symptoms associated with schizophrenia can be reproduced by administering NMDA antagonists to healthy subjects, and symptom severity in patients are exacerbated following exposure to these agents (Farber, 2003, Javitt and Zukin, 1991, Jentsch and Roth, 1999, Krystal et al., 1994, Lahti et al., 1995, Malhotra et al., 1997, Malhotra et al., 1996). While the psychotomimetic effects of NMDA antagonists were originally emphasized with respect to positive symptoms, much evidence in both laboratory animals and humans now suggests that NMDA antagonism may also contribute to the development of both negative symptoms and cognitive dysfunction (Honey et al., 2003, Honey et al., 2005, Krystal et al., 1994, Malhotra et al., 1996). Such findings have led to a focus on the development of pharmacological methods that model these aspects of schizophrenia symptomology in laboratory animals. To date, several laboratories have demonstrated schizophrenia-like changes on a number of behavioral measures relevant to positive, negative, and cognitive symptoms in laboratory animals exposed to chronic NMDA antagonist administration (Ketamine, PCP or MK801) (Chan et al., 2008, Featherstone et al., 2012, Jentsch and Roth, 1999, Kittelberger et al., 2012, Lazarewicz et al., 2010, Neill et al., 2010, Rushforth et al., 2011, Saunders et al., 2012a, Siegel et al., 2003).
The development of animal models of hypoglutamate function demonstrating stable and reliable changes in behaviors relevant to schizophrenia will be crucial for the development of new approaches for drug discovery in schizophrenia. To this end, we recently assessed a series of electrophysiological and behavioral responses in a transgenic mouse (NR1−/−) that shows only 5–10% normal expression of the NR1 subunit of the NMDA receptor (Bodarky et al., 2009, Gandal et al., 2012a, Gandal et al., 2012b, Halene et al., 2009, Mohn et al., 1999). These NMDAR NR1 deficient mice show disrupted sensory processing, as assessed by auditory event-related potentials (ERPs) or pre-pulse inhibition, reduced behavioral inhibition, and impaired social interaction (Bodarky et al., 2009, Duncan et al., 2004, Duncan et al., 2006, Duncan et al., 2012, Gandal et al., 2012b, Halene et al., 2009, Mohn et al., 1999, Moy et al., 2006). Additionally, NMDAR NR1 deficient mice demonstrate increased resting, and reduced high frequency (gamma, 30–100 Hz) EEG oscillations in response to auditory stimulation (Gandal et al., 2012b). Reduced evoked gamma power has also been widely reported in schizophrenia (Roach and Mathalon, 2008, Spencer et al., 2008, Spencer et al., 2009, Uhlhaas and Singer, 2010), and it has been suggested that this deficit may be the physiological basis for or signifier of impairments observed in schizophrenia. The use of EEG measures such as ERPs and gamma oscillations are important for translational studies since they can easily be measured in both humans and laboratory animals, and because the response of these measures to pharmacological agents and variation in stimulation parameters, including inter-stimulus interval, stimulus intensity, sensory adaptation (a.k.a. gating), and stimulus novelty, all of which show a high degree of similarity across species (Amann et al., 2010, Siegel et al., 2013).
Despite such positive findings, full NR1 hypomorphs may present too severe a phenotype to be relevant for schizophrenia. Homozygous hypomorphs show up to 95% reduced expression of the NMDA NR1 subunit (Mohn et al., 1999), a reduction much greater than what has been shown in post-mortem studies of schizophrenia brains (Gao et al., 2000, Weickert et al., 2013). Additionally, full hypomorphs show phenotypic changes not specific to schizophrenia (Gandal et al., 2012a). In contrast to the full knockdown, we propose that heterozygous (NR1+/−) mice show reductions in NR1 receptor levels that are more consistent with the degree of subtle NMDAR-mediated impairments in glutamate transmission in the human disease. The current study sought to assess the validity of NR1 heterozygous knockout mice as a translational model for subtle reduction in NMDAR function that has been proposed in schizophrenia using EEG and ERP measures that have been well-established for detecting schizophrenia-like changes. Additionally, the additive effects of genetic vulnerability (NR1+/−) and environmental stress (social isolation) were assessed in order to determine the validity of these mice using a two-hit model of schizophrenia vulnerability. Social isolation stress has been associated with the development of schizophrenia-like symptoms both in human populations and laboratory animals (Fone and Porkess, 2008, Geyer et al., 1993, Hodges et al., 1999). Further, the detrimental effects of social isolation are enhanced in rodents with reduced NMDA receptor function (Belforte et al., 2010, Gaskin et al., 2014, Jiang et al., 2013), suggesting that increased susceptibility to environmental insults in patients with schizophrenia might reflect reduced glutamate function. The establishment of a two-hit model combining genetic and environmental insults would be extremely useful for the development of treatments to prevent the development of symptom onset in genetically vulnerable individuals. Given the available evidence, it was predicted that heterozygous NR1 (+/−) mice with reduced glutamate function would be more sensitive to the disruptive effects of social isolation.
Section snippets
Subjects
NR1neo+/− mice (MGI:95819) (Mohn et al., 1999, Duncan et al., 2004, Bickel, 2008) were in-licensed from the laboratory of Dr. Beverly Koller (The University of North Carolina at Chapel Hill) and a breeding colony was established by Astellas Research Institute of America (Skokie, Illinois), maintained at Hilltop Lab Animals (Scottsdale, PA). Mice were maintained on C57BL/6NHla background. The PCR protocol used for genotyping these mice was as follows: NR1 (+) fwd primer (intron 20) 5′TGA GGG GAA
qPCR for NR1 receptor expression
Data were analyzed using a Factorial ANOVA with genotype (NR1 +/− or WT), housing condition (group or isolation) and brain region (DG and PFC) as independent variables and expression level as the dependent variable. NR1 levels were significantly lower in (NR1+/−) relative to wild-type mice (F = 126.3, p < 0.001). There was no main effect of rearing condition, nor was there an interaction between genotype and rearing condition in either brain region. However, there was a significant effect of brain
Discussion
Neither genotype nor rearing condition produced significant reductions in P20 and N40 ERP amplitude in the current study, suggesting that these measures are not sensitive to subtle reductions in NMDA receptor function. The lack of effect on either S1 amplitude on its attenuation is consistent with the finding that disruptions in these measures are typically observed only following relatively high doses of NMDA antagonists (de Bruin et al., 1999, Lazarewicz et al., 2010, Oranje et al., 2002,
Summary and conclusion
The observation that (NR1+/−) mice with subtle reduction in expression of NR1 showed an exclusive deficit on MMN suggests that this measure can be used to detect subtle alterations in glutamate function that are not detected using alternate EEG measures. As such, the MMN may be an important biomarker of early alterations, as may occur during the prodromal period (Perez et al., 2014).
Acknowledgment
Funding provided by 5R01DA023210-02 (SJS) and an investigator initiated research grant from Astellas (SJS) (10031929). J. Kogan, R. Shin and M. Matsumoto are employees of Astellas Research Institute of America.
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