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