Social isolation rearing induces mitochondrial, immunological, neurochemical and behavioural deficits in rats, and is reversed by clozapine or N-acetyl cysteine

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Abstract

Apart from altered dopamine (DA) function, schizophrenia displays mitochondrial and immune-inflammatory abnormalities, evidenced by oxidative stress, altered kynurenine metabolism and cytokine release. N-acetyl cysteine (NAC), an antioxidant and glutamate modulator, is effective in the adjunctive treatment of schizophrenia. Social isolation rearing (SIR) in rats is a valid neurodevelopmental animal model of schizophrenia. This study evaluated whether SIR-induced behavioural deficits may be explained by altered plasma pro- and anti-inflammatory cytokines, kynurenine metabolism, and cortico-striatal DA and mitochondrial function (via adenosine triphosphate (ATP) release), and if clozapine or NAC (alone and in combination) reverses these changes. SIR induced pronounced deficits in social interactive behaviours, object recognition memory, and prepulse inhibition (PPI), while simultaneously increasing striatal but reducing frontal cortical accumulation of ATP as well as DA. SIR increased pro- vs. anti-inflammatory cytokine balance and altered kynurenine metabolism with a decrease in neuroprotective ratio. Clozapine (5 mg/kg/day × 14 days) as well as clozapine + NAC (5 mg/kg/day and 150 mg/kg/day × 14 days) reversed these changes, with NAC (150 mg/kg/day) alone significantly but partially effective in some parameters. Clozapine + NAC was more effective than clozapine alone in reversing SIR-induced PPI, mitochondrial, immune and DA changes. In conclusion, SIR induces mitochondrial and immune-inflammatory changes that underlie cortico-striatal DA perturbations and subsequent behavioural deficits, and responds to treatment with clozapine or NAC, with an additive effect following combination treatment. The data provides insight into the mechanisms that might underlie the utility of NAC as an adjunctive treatment in schizophrenia.

Highlight

► Social isolation rearing induces mitochondrial, immunological, dopaminergic and behavioural deficits akin to schizophrenia, and reversed by clozapine or N-acetyl cysteine.

Introduction

Schizophrenia is characterised by positive, negative and cognitive symptoms (Schultz et al., 2007), with evidence implicating genetic predisposition, neurotransmitter abnormalities, pre-, peri- and post-natal viral infections, stress, substance abuse, vitamin D deficiency, obstetric complications and altered immune function in its pathogenesis (O’Brien et al., 2008, McGrath et al., 2010, Matheson et al., 2011). The dopamine (DA) hypothesis suggests that schizophrenia is causally associated with elevated striatal (nucleus accumbens) but reduced frontal cortical DA levels responsible for positive and negative manifestations, respectively (Harvey et al., 1999, Holcomb et al., 2004). Current drug treatments, which primarily act at DA D2/3 receptors, are generally effective against positive symptoms, while negative and cognitive symptoms remain relatively refractory (Howes et al., 2012). Identifying new biological targets and developing treatments targeting alternative systems is therefore paramount.

Schizophrenia is associated with mitochondrial dysfunction (Ben-Shachar, 2002, Karry et al., 2004) and oxidative stress (Ng et al., 2008). Indeed, altered energy generation is perhaps the oldest biomarker in the disorder (Looney and Childs, 1934), while altered frontal lobe mitochondrial adenosine triphosphate (ATP) has been correlated with negative symptoms and neuropsychological deficits in schizophrenia (Yacubian et al., 2002). Mitochondria are a major source of reactive oxygen species (ROS), while mitochondrial diseases are associated with secondary neurotransmitter disturbances (Garcia-Cazorla et al., 2008). This is of particular relevance in schizophrenia where disturbances in DA are evident (Ben-Shachar, 2002). Moreover, mitochondrial dysfunction is linked to cognitive and memory deficits, evident in schizophrenia (Scaglia, 2010). Importantly elevated ATP is also a trigger for the release of pro-inflammatory cytokines (Cruz et al., 2007), which in turn modulates kynurenine metabolism (see below). Thus schizophrenia presents with a pro-inflammatory state (Leonard et al., 2012), with altered blood/cerebrospinal fluid levels of interleukin-2 (IL-2), IL-6, IL-1, IL-10 and tumour necrosis factor (TNF)-α (Drzyzga et al., 2006). Mitochondrial ATP also modulates glial cytokine release, while up-regulation in DA receptors play an important role in this response (Choi et al., 2007). With DA release closely regulated by glutamate (Schwartz et al., 2012), any glutamate-redox changes will alter cortico-striatal DA function to ultimately affect behaviour.

The kynurenine pathway metabolises tryptophan via indoleamine 2,3 dioxygenase (IDO) to either kynurenic acid (KYNA), a glycine site NMDA receptor antagonist with neuroprotective properties (Stone and Darlington, 2002), or quinolinic acid (QA), an NMDA receptor agonist with neurodegenerative properties (Myint et al., 2007). Pro- (e.g. interferon-γ (INF-γ), TNF-α) and anti-inflammatory (e.g. IL-4) cytokines, respectively, activate or inhibit IDO (Myint et al., 2007). This balance determines the neuroprotective-neurodegenerative ratio that relates to the eventual downstream levels of QA following a shift in KYNA and kynurenine synthesis (Myint et al., 2007, Myint et al., 2011), and may explain enlarged ventricles (Wright et al., 2000) and decreased dendritic spine density (Glantz and Lewis, 2000) evident in schizophrenia (Wright et al., 2000). Schizophrenia presents with altered kynurenine metabolism (Miller et al., 2004, Miller et al., 2008), increased QA and decreased neuroprotective ratio (Torrey et al., 1998, Myint et al., 2011), with subsequent NMDA receptor activation releasing ROS and depleting energy stores resulting in further glutamate release and cell damage (Betzen et al., 2009). Glutamate dysfunction will also drive changes in DA release culminating in positive and negative symptoms (Schwartz et al., 2012).

It is clear that oxidative stress can be a consequence of mitochondrial dysfunction, brain DA metabolism, immune dysregulation and/or altered tryptophan metabolism. Consequently, targeting redox related mechanisms in schizophrenia represents an important therapeutic target (Ng et al., 2008, Cabungcal et al., 2012). The glutathione (GSH) precursor and antioxidant, N-acetyl cysteine (NAC) modulates glutamate activity (Bauzo et al., 2012), decreases ROS production (Kerksick and Willoughby, 2005) and prevents striatal oxidative stress in vivo (Harvey et al., 2008). Furthermore, remediation of models of mitochondrial dysfunction with NAC improves cognitive function (Sandhir et al., 2012). NAC thus presents with significant theoretical rationale for clinical application in disorders characterised by glutamate dysregulation, mitochondrial function, inflammation and oxidative stress. Although clinical studies have described its potential as an adjunctive treatment in schizophrenia (Berk et al., 2008), this response has never been validated in a neurodevelopmental animal model of schizophrenia, especially its ability to correct behavioural and biochemical changes and to bolster the response to an antipsychotic agent. In particular, knowing how these biological pathways are linked to neurobehavioural phenotypes might shed some light onto the dominant mechanisms of action of NAC, and suggest further therapeutic targets.

Post-weaning social isolation rearing (SIR) in rodents presents with robust face, construct and predictive validity for schizophrenia (Fone and Porkess, 2008). SIR increases frontal cortical glutamate N-methyl-d-aspartate (NMDA) receptor binding (Toua et al., 2010) and induces cortico-striatal oxidative stress (Möller et al., 2011), thus supportive of clinical evidence and of the glutamate hypo-function hypothesis (Coyle and Tsai, 2004). We hypothesise that the behavioural abnormalities associated with schizophrenia and SIR are founded on altered cortico-striatal mitochondrial dysfunction, followed by altered plasma pro-and anti-inflammatory cytokines and tryptophan metabolism, with subsequent changes in regional brain DA. This series of events is investigated in this study. Furthermore, we propose that sub-chronic clozapine or NAC treatment will reverse these changes, and that NAC will bolster the response to clozapine.

Section snippets

Animals

Male Sprague-Dawley rats (160–190 g; Animal Research Centre, North West University) were randomly allocated to groups, each comprising 10 rats/group. At weaning (post-natal day 21) the animals were randomised to SIR (1 animal/cage) or social rearing (3–4 rats/cage) for 8 weeks (day 77). The rats were reared under identical conditions: cages (230(h) × 380(w) × 380(l) mm) with sawdust (Möller et al., 2011), temperature (21 ± 0.5 °C), humidity (50 ± 10%), white light (350–400 lux), 12 h light/dark cycle and free

Results

In the drug treatment response studies, socially reared animals showed minimal changes across all parameters, and these data are presented in Supplement 1 (Fig. 1, Fig. 2, Fig. 4, Fig. 5, Fig. 6, Fig. 7). Only “social plus vehicle treatment” data are presented in the figures for statistical comparison. Unless specifically noted, statistical analysis of vehicle-treated vs. non-treated SIR groups was not significant, with all subsequent inter-group comparisons done vs. vehicle-treated SIR.

Discussion

This study has reaffirmed the validity of SIR in rats as a model for schizophrenia, including the reduction of %PPI (Fig. 3; Geyer et al., 1993), effects on social interactive behaviours (Fig. 1; Möller et al., 2011), elevated kynurenine and QA vs. KYNA and reduced neuroprotective ratio (Fig. 5; Möller et al., 2012b), and their reversal by clozapine (Möller et al., 2011, Möller et al., 2012b). We now show that SIR reduces plasma IL-4 and IL-6 (Fig. 4A and B) and increases TNF-α and IFN-γ (Fig. 4

Conclusion

SIR-induced behavioural deficits in social interaction, memory and PPI are associated with altered cortico-striatal ATP levels, an increase and decrease in pro- and anti-inflammatory cytokines, respectively, leading to altered plasma tryptophan metabolites, a reduction in the neuroprotective ratio and cell damage. These effects combine to initiate striatal DA changes expressed as positive- or negative-like behavioural symptoms. Moreover, these changes are reversed by sub-chronic clozapine or

Author disclosures

Sources of funding: The authors declare that this work has been funded by the South African Medical Research Council (BHH). The funder has no other role in this study.

Conflicts of interest: The authors declare that over the past three years, Robin Emsley has participated in speakers/advisory boards and received honoraria from AstraZeneca, Bristol-Myers Squibb, Janssen, Lilly, Lundbeck, Organon, Pfizer, Servier, Otsuka and Wyeth. He has received research funding from Janssen, Lundbeck and

Acknowledgments

The authors would like to thank Mr. Cor Bester, Me. Antoinette Fick and Mr. Petri Bronkhorst for their assistance in the breeding and welfare of the animals.

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