Changes in cholinergic and glutamatergic markers in the striatum from a sub-set of subjects with schizophrenia

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Abstract

Background/objectives

Having separated a sub-group of people with schizophrenia based on a marked loss of cortical [3H]pirenzepine binding (MRDS); we wished to determine if MRDS had lower levels of [3H]pirenzepine and other muscarinic receptor antagonist binding to the striatum and if this was due to loss of pre- or post-synaptic neurons or glia measured using surrogate markers (25 kilodalton synaptosomal-associated protein (SNAP 25), postsynaptic density protein 95 (PSD 95), glial fibrillary acidic protein (GFAP) 41/43) of cell number.

Methods

[3H]pirenzepine, [3H]AF-DX 384 and [3H]4-DAMP binding to the striatum from 37 subjects with schizophrenia (19 MRDS) and 20 controls as well as SNAP 25, PSD 95 and GFAP 41/43 in crude particulate membrane were measured.

Results

[3H]pirenzepine and [3H]AF-DX 384 binding to the striatum were significantly lower in schizophrenia due to lower binding of both radioligands in the striatum from MRDS. Levels of PSD 95 were higher in schizophrenia, predominantly due to higher levels in MRDS.

Conclusions

Our data suggest muscarinic M1 ([3H]pirenzepine) and M2 and/or M4 receptors ([3H]AF-DX 384) are lower in the striatum from MRDS which could mediate inappropriate adaption to internal and external cues which, in turn, would affect motivation, cognition and motor control. Increased levels of PSD 95 could indicate increased post-synaptic boutons or changes in NMDA receptor-mediated signalling in MRDS.

Introduction

Treatment resistance remains a significant problem in the management of schizophrenia. Recent evidence suggests that targeting specific muscarinic receptors (CHRMs) with agonists (Shekhar et al., 2008) or allosteric modulators (Conn et al., 2009) can reduce the severity of symptoms associated with the disorder. This proposed approach, which involves stimulating the cholinergic system in the CNS of subjects with schizophrenia, is relevant to the reproducible finding of lower levels of CHRMs in the CNS of subjects with schizophrenia (Raedler et al., 2007) as low levels of these receptors are suggestive of an underactive cholinergic system. Studies using post-mortem tissue have reported lower levels of [3H]pirenzepine binding to CHRMs in the striatum, cortex, hippocampus and thalamus from subjects with schizophrenia (for Review see (Scarr et al., 2013b)). Importantly, under the conditions most used to measure [3H]pirenzepine binding the radioligand is highly selective for the muscarinic M1 receptor (CHRM1) (Gibbons et al., 2012, Scarr and Dean, 2008) and hence it is likely there are low levels CHRM1 in the CNS from subjects with schizophrenia. This proposition is supported by data showing decreased cortical CHRM1 mRNA (Dean et al., 2002, Mancama et al., 2003) and protein (Dean et al., 2002) in subjects with the disorder.

A significant hindrance to understanding the underlying aetiology of schizophrenia is that the current symptom-based diagnosis defines a syndrome of disorders (Kirkpatrick et al., 2001). We have suggested that the loss of [3H]pirenzepine binding in the dorsolateral prefrontal cortex (Brodmann's area 9) is limited to a distinct sub-group (25%) of subjects with schizophrenia and we termed this sub-group muscarinic receptor deficit schizophrenia (MRDS) (Scarr et al., 2009). Our more recent data shows that subjects with MRDS have changes in cortical agonist-induced recruitment of G-protein by the CHRM1 (Salah-Uddin et al., 2009), methylation patterns on the CHRM1 gene promoter (Scarr et al., 2013a), increased levels of miR-107 (Scarr et al., 2013a) and lower levels of [3H]pirenzepine binding in multiple cortical regions (Gibbons et al., 2012). The generalised loss in [3H]pirenzepine binding across the cortex is consistent with the widespread losses of CHRMs in the CNS from subjects with schizophrenia measured using neuroimaging (Raedler et al., 2003).

In the striatum we have reported lower [3H]pirenzepine binding and no change in the levels of CHRM1 mRNA (Dean et al., 2000). We have also reported lower levels of [3H]AF-DX 384, a radioligand with highest affinity for the CHRM2 and M4 (Miller et al., 1991), in the striatum from subjects with schizophrenia (Crook et al., 1999). These data suggest that, unlike in the cortex, changes in the levels of multiple CHRMs may be occurring in the striatum from subjects with schizophrenia. Hence, here we extend our studies beyond the cortex to measure [3H]pirenzepine, [3H]AF-DX 384 and [3H]4DAMP (CHRM3 (Jeon et al., 2013)) binding to the striatum from subjects with schizophrenia and age sex matched controls; the cohort with schizophrenia consisted of 19 subjects with MRDS and 18 subjects that we designate as non-MRDS from our study in the human cortex (Scarr et al., 2009). Further, in the same subjects we have measured levels of surrogate markers of pre- (25 kilodalton synaptosomal-associated protein (SNAP 25)) and post-synaptic (postsynaptic density protein 95 (PSD 95)) (Gylys et al., 2004) neurons and astrocytic ((GFAP) 41 and 43) number (Sofroniew and Vinters, 2010).

Section snippets

Ethical approval

Approvals for these studies were from the Ethics Committee of the Victorian Institute of Forensic Medicine and the Tissue Access Committee of the Victorian Brain Bank Network.

Tissue collection

All human tissues were sourced through the Victorian Brain Bank Network at the Florey Institute for Neuroscience and Mental Health, Parkville, Australia.

Tissue was initially collected from subjects with a potential history of a psychiatric disorder and subjects with no apparent history of psychiatric disorders who were

Demographic, pharmacological and CNS collection

There were no significant differences in age, sex ratio, PMI or CNS pH with diagnoses (Table 1, for full details see Supplementary Table 1). There was a significant difference in the number of suicide completers with diagnoses as completers were only in the group with schizophrenia. Similarly, there were no significant differences in age, sex ratio, PMI and CNS pH between MRDS, non-MRDS and controls but the frequency of suicide completion differed across groups (Table 1). Finally, there were no

Discussion

The major finding from our studies is that, compared to controls, there are lower levels of [3H]pirenzepine and [3H]AF-DX 384 binding to the striatum from subjects with schizophrenia. Importantly, the lower levels of [3H]pirenzepine and [3H]AF-DX 384 binding in schizophrenia are restricted to the striatum from subjects with MRDS. Levels of [3H]4DAMP binding are not changed in schizophrenia, subjects with MRDS or subjects with non-MRDS. Significantly, the acetylcholine that activates cortical

Contributors

Author BD was involved in formulating the design of the study, completed the statistical analyses and produced the first draught of the manuscript. Author NT wrote the protocols for radioligand binding, produced the data from that methodology and was involved in editing the manuscript to its final form. Author CY-L wrote the protocols for Western blotting, produced the data using that analyses was involved in editing the manuscript to its final form. Authors WJC was involved in the study design

Funding sources

This research was funded by the NHMRC, the Victorian Government's Operational Infrastructure Support and the Rebecca Cooper Medical Research Foundation, none of which had any influence over the generation of data or the writing of the submitted manuscript.

Conflicts of interest

The authors have no conflicts of interest regarding this manuscript.

Acknowledgements

BD is an NHMRC Senior Research Fellow (APP1002240). This work was supported in part by NHMRC project grants (APP1045619 and APP628699), the Victorian Government's Operational Infrastructure Support and the Rebecca Cooper Medical Research Foundation.

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