NeuN+ neuronal nuclei in non-human primate prefrontal cortex and subcortical white matter after clozapine exposure

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Abstract

Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved.

We exposed 26 young adult macaque monkeys for 6 months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts.

After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups.

Chronic clozapine exposure increases the proportion of NeuN+ nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted.

Introduction

Schizophrenia, a major psychiatric disorder significantly impacting quality of life, is commonly treated with antipsychotic drugs but many patients show insufficient responses to current treatments (Lieberman et al., 2005, Swartz et al., 2007). Therefore, the pursuit of new schizophrenia treatments should start, among other approaches, with detailed explorations of transcriptomes (Feher et al., 2005, Girgenti et al., 2010, Iancu et al., 2012, Middleton et al., 2002) and synaptic proteomes (Ji et al., 2009, Ma et al., 2009) in the brains exposed to typical dopamine D2-preferential antagonists and atypical antipsychotic drugs with broader receptor profiles. Changes in cell composition of the antipsychotic-drug-exposed brain have also been reported. Long-term exposure of non-human primates to two widely prescribed antipsychotics, haloperidol and olanzapine, resulted in 8–11% brain weight reduction and volume loss affecting gray and white matter, decreased astro- and oligodendrocyte numbers (Konopaske et al., 2008), together with a 10.2% increase in neuronal densities (Konopaske et al., 2007).

These findings are also of interesting from the viewpoint of the interstitial white matter neurons (WMN), a cell type residing in subcortical white matter of the adult brain. The large majority of WMN are considered remnants of the subplate, a transient structure important for connectivity formation during early development (Kanold, 2004, Kostovic et al., 2011). Interestingly, more than 15 studies have examined post-mortem brain tissue and reported supernormal WMN numbers and densities in prefrontal, cingulate and medial or lateral temporal cortex of subjects diagnosed with schizophrenia (Akbarian et al., 1993a, Akbarian et al., 1996, Akbarian et al., 1993b, Anderson et al., 1996, Eastwood and Harrison, 2003, Eastwood and Harrison, 2005, Ikeda et al., 2004, Joshi et al., 2012, Kirkpatrick et al., 1999, Kirkpatrick et al., 2003, Rioux et al., 2003, Yang et al., 2011). While negative findings have also been published (Beasley et al., 2002, Beasley et al., 2009) most research on this topic indicates that WMN alterations could affect a subset of up to 25% of patients with schizophrenia (Connor et al., 2009). It remains unclear whether increased numbers of WMN in patients with schizophrenia marks a subtype of schizophrenia and if treatment with antipsychotic medication in vivo plays any role. So far, medication-induced changes in WMN never have been explored in a controlled, prospective study. This is both surprising, given the potential importance of antipsychotic drugs, which are widely prescribed to millions of patients, and anticipated, as controlled studies on drug-mediated effects very difficult to conduct on human brain.

Here, we designed an integrative study on 26 macaque monkeys subjected to 6 months of oral intake of haloperidol and clozapine, followed by MRI-based in vivo neuroimaging with scans before and after antipsychotic drug exposure, followed by automated quantification of neuron to glia (non-neuron) ratios in frontal gray and white matter and cell-type specific molecular fingerprinting (Fig. 1). We choose haloperidol and clozapine because these drugs are extensively prescribed prototypes representing conventional antipsychotics primarily acting as dopamine D2 receptor antagonists (haloperidol) or atypical antipsychotics with broader receptor profiles (clozapine). Furthermore, clozapine is generally considered of superior therapeutic efficiency compared to many of the typical or atypical antipsychotics (Meltzer, 2013, Wenthur and Lindsley, 2013). We report increased proportions of nuclei expressing the neuronal phenotypic marker for ‘Neuronal Nuclei’ (NeuN) (Mullen et al., 1992) after clozapine exposure, affecting subcortical white matter and more subtle changes in overlying cortex, without affecting cortical volumes or cell type specific gene expression.

Section snippets

Animals and antipsychotic drug treatments

26 young adult and drug-naïve rhesus macaques (12 female, 14 male) were randomly assigned to one of the three treatment groups: haloperidol (4 mg/kg/day), clozapine (5.2 mg/kg/day), or vehicle (Table 1). Using previously established protocols (Lidow et al., 1997, Lidow and Goldman-Rakic, 1994, Lidow and Goldman-Rakic, 1997), monkeys were administered antipsychotic drugs orally for six months, mixed with powdered sugar and given in peanut butter or fruit treats. Monkeys received standard

Results

Monkeys were subjected to in vivo neuroimaging at two different time points before and then again 5.5 months after begin of antipsychotic treatment, prior to brain harvest and tissue biopsy for flow cytometry-based neuronal and non-neuronal quantification and, for a subset of brains, cell-type specific RNA analysis (Fig. 1). The daily haloperidol dose (4 mg/kg/day) resulted in plasma steady state trough levels approaching 2 ng/ml (Table 1). In patients with schizophrenia, trough levels as low as

Discussion

In this non-human primate study, clozapine exposure was associated with a significantly (~ 50%) increased proportion of NeuN+ nuclei in frontal subcortical white matter. This was correlated with a much milder elevation of NeuN+ proportions in the overlying gray matter, which could reflect common regulatory mechanisms across the two anatomical compartments. These alterations were highly specific, because OLIG2+ nuclei, representing the oligodendrocyte lineage(Yokoo et al., 2004) and in complete

Funding and disclosure

This study was supported by Brain Behavior Research Foundation (Distinguished Investigator award to S.A.), National Institutes of Health grants MH074313 and a research grant from the Medical Research Institute. The authors have no conflict of interest to declare.

Contributors

Schahram Akbarian and Scott Hemby developed the idea and wrote this article with Tobias Halene who was also responsible for flow-cytometry (with Alexey Kozlenkov and Stella Dracheva and Royce Park and Jennifer Wiseman), MRI image processing and analysis (with Paula Croxsom) and RT-PCR (with Amanda Mitchell and Yan Jiang. Aslihan Dincer provided RNA-seq tracks, Patrick Hof and also Behnam Javidfar conducted immunohistochemical experiments and provided immunofluorescence confocal microscopy

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgments

The authors thank Brian Horman, Jim Daunais, Yin Guo, Lily C. Lin, and the staff from the Wake Forest University Primate Center for excellent technical support.

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    Present address: Department of Basic Pharmaceutical Sciences, Highpoint University, 833 Montlieu Ave, NC 27262, USA.

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