Evidence for altered post-receptor modulation of the serotonin 2a receptor in schizophrenia
Introduction
A large body of data suggests there is a decrease in serotonin 2A receptors (HTR2A) in the CNS of subjects with schizophrenia (Dean, 2003). Although two neuroimaging studies, using positron emission tomography, support this hypothesis (Ngan et al., 2000, Okubo et al., 2000), a greater number of such studies have failed to observe a change in HTR2A availability in subjects with the disorder (Lewis et al., 1999, Trichard et al., 1998, Verhoeff et al., 2000). However, it has recently been reported that decreased levels of HTR2A are present in the CNS of subjects with an enhanced risk of developing schizophrenia (Hurlemann et al., 2005), suggesting that low levels of HTR2A may be present early in the course of the illness. This finding suggests that decreased HTR2A may be a biomarker for an increased risk of schizophrenia; therefore it is imperative to resolve the apparent discrepancies as to whether there are changes in HTR2A in subjects with the disorder.
The majority of studies on HTR2 receptors in schizophrenia have been completed using postmortem CNS. These studies were originally conducted using tissue homogenates and as is the case with neuroimaging, mainly report that there was no change in HTR2A in the CNS of subjects with schizophrenia (Dean and Hayes, 1996, Eastwood et al., 2001, Laruelle et al., 1993, Reynolds et al., 1983b, Whitaker et al., 1981); by contrast, two studies reported levels of the receptor were decreased in subjects with the disorder (Arora and Meltzer, 1991, Mita et al., 1986). These discrepancies were accentuated with the development of in situ radioligand binding where most studies reported a decrease in HTR2A in the CNS from subjects with schizophrenia (Burnet et al., 1996, Dean et al., 1999, Dean and Hayes, 1996, Gurevich and Joyce, 1997, Matsumoto et al., 2005, Pralong et al., 2000), with only one report suggesting levels of HTR2A were not altered (Joyce et al., 1993). This significant change in the ratio of negative to positive findings on HTR2A with different techniques clearly suggests that methodological factors might result in different outcomes using postmortem CNS from subjects with schizophrenia.
There are a number of methodological factors that could cause apparent differences in outcomes when measuring HTR2A in postmortem CNS from subjects with schizophrenia. However, using the same combination of radioligand and displacing agent our studies show either no change (Dean and Hayes, 1996) or decreased (Dean and Hayes, 1996) levels of HTR2A in tissue samples from the same individuals using homogenates and in situ radioligand binding with autoradiography, respectively. This suggests that changes in HTR2A in schizophrenia are not simply a result of using tissue from different cohorts or the selection of radioligand and displacer. Similarly, our data using tissue from the same individuals suggest that differences in residual antipsychotic drug levels are unlikely to be the cause of variation in data from different methodologies. Moreover, in our homogenate binding, the affinity of radioligand binding did not vary between subjects with schizophrenia and the control subjects when analyzed by Scatchard plots (Dean et al., 1996). Importantly, some antipsychotic drugs can act as competitive antagonists at the HTR2A (Bymaster et al., 1996) and could therefore be a confound in the study of HTR2A in schizophrenia. However, the binding of [3H]ketanserin would also be affected by the presence of such drugs in postmortem tissue and we failed to show any change in that measure across disease cohorts in our membrane binding study (Dean et al., 1997). Thus, it seemed that the different outcomes from the measurement of the HTR2A receptor in schizophrenia were not likely to be simply due to residual antipsychotic drugs.
It has been widely reported that treating rats with antipsychotic drugs that have an affinity for the HTR2A reduces levels of the receptor in the cortex (Dean, 2003). This raises the possibility that decreases in the HTR2A in postmortem tissue from subjects with schizophrenia could have resulted from treatment with antipsychotic drugs which target that receptor. However, if such affects are rapidly reversible that would suggest that their should be differences in radioligand binding between subjects that died while being treated with antipsychotic drugs compared to those who ceased treatment some time before death.
Our existing data suggests that some of the differences in the studies on 5HTR2A in postmortem CNS from subjects with schizophrenia could be due to the use of different methodologies. The major difference between radioligand binding studies using membrane preparations and those using tissue sections is that, in the case of the tissue section, the membrane an intra-cellular milieu remains in tack. Thus, to better understand the problems relating to findings on HT2AR in postmortem CNS, we first measured levels of HTR2A using single-point saturation analysis and tissue sections, crude tissue homogenate (in which the membrane is left in contact with the intra-cellular milieu), membrane-enriched (ME; where the membrane is removed from the intra-cellular milieu) fractions and cytosol-enriched (CE; which is the intra-cellular milieu alone) tissue fractions from Brodmann's area (BA) 9. These measurements were made by preparing all tissue sections and preparations sequentially from the same tissue block to determine whether tissue processing might influence the outcomes of studying HT2AR in the cortex of subjects with schizophrenia. Second, we measured the impact of adding antipsychotic drugs to all these preparations of human CNS from control subjects to determine if a particular tissue preparation may be susceptible to the presence of such drugs when measuring levels of HTR2A. Finally, we assessed the potential impact of antipsychotic drug treatment in rat cortex and whether these effects could be reversed on cessation of drug treatment. The reversibility of antipsychotic drug effects is important because of i) the high rate of medication non-compliance in subjects with schizophrenia (Byerly et al., 2007) and ii) the unexpectedly low levels of HTR2A in the cortex of subjects with schizophrenia who had been recorded as being prescribed medication close to death (Rodda et al., 2006).
Section snippets
Human CNS and preparation
The study of human postmortem tissue was undertaken following consent by the Ethics Committees of the Victorian Institute of Forensic Medicine and the North Western Mental Health Program Behavioural and Psychiatric Research and Ethics Committee.
Tissue was collected from BA 9 from 14 subjects with schizophrenia and 14 subjects with no history of psychiatric or neurological disorders (controls) closely matched for sex and matched for age to the subjects with schizophrenia. Cortical tissue
Results
There were no significant differences in the mean age (p = 0.95), PMI (p = 0.82) or CNS pH (p = 0.65) between the control subjects and subjects with schizophrenia. There was no difference in the sex ratio between the diagnostic cohorts (p = 1.00).
The level of [3H]ketanserin binding to the inner layer of the cortex from control subject #13 and ME tissue from control subject #6 (Table 1) were shown to be statistically significant outliers and excluded from subsequent analyses. All radioligand binding
Discussion
Our data shows that the ability to detect differences in [3H]ketanserin binding in postmortem CNS from subjects with schizophrenia is dependent on maintaining the association between membrane and cytosol within the tissue. This phenomena means our study is in agreement with both studies using tissue sections that show decreased levels of HTR2A in the CNS of subjects with schizophrenia (Burnet et al., 1996, Dean et al., 1998, Dean et al., 1999, Dean and Hayes, 1996, Gurevich and Joyce, 1997,
Role of funding source
Funding for this study was provided by NHMRC, The Australian Rotary Health Research Fund and The Rebecca L. Cooper Medical Research Foundation. None of the funding bodies had any further role in the study design; in the collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the paper for publication.
Contributors
Author Brian Dean was involved in the design of the study, undertook data analysis and wrote the manuscript. Author Nicola Crossland was involved with the design of the study, completed experimental protocols and assisted by editing drafts of the manuscript. Author Simone Boer was involved in the design of the study, completed the experimental protocols and assisted by editing each draft of the manuscript. Author Elizabeth Scarr was involved in the design of the study and in editing all drafts
Conflict of interest
The authors have no conflicts of interest.
Acknowledgments
BD is a NHMRC Senior Research Fellowship (# 400016) and ES is the Ronald Griffiths Schizophrenia Research Fellow and holds a NARSAD Young Investigator Award. The study was also supported by NHMRC Project Grant # 114253, NIMH Grant # MH069691-01A1, The Rebecca L. Cooper Medical Research Foundation.
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