The distribution of muscarinic M1 receptors in the human hippocampus
Introduction
The role of the cholinergic system in cognitive processes, such as attention and information processing, is well established (Carruthers et al., 2015). Importantly, it has been shown that muscarinic and nicotinic receptors play synergistic roles in controlling cognitive processes such as working memory (Ellis et al., 2006) with nicotinic receptors modulating phasic activity important in detecting cues and muscarinic receptors controlling tonic activity which is important for attentional control (Recent review: (Demeter and Sarter, 2013)). In addition, the cholinergic system within the septohippocampal pathway has been suggested to play a role in acquisition related to short term memory and recognition (Klinkenberg et al., 2011). Data from muscarinic M1 receptor knock out mice (Chrm1−/−) suggests that the muscarinic M1 receptor has a critical role in memory processes occurring in the hippocampus (Anagnostaras et al., 2003), reinforcing the role of the hippocampal muscarinic M1 receptor in maintaining aspects of cognitive abilities in humans and other mammals.
Until recently, the high degree of structural homology at the orthosteric binding site on the five muscarinic receptors has meant it has not been possible to develop drugs specific for individual muscarinic receptors (Kruse et al., 2014). However, drugs have now been synthesised that are highly selective for, if not specific to, each individual muscarinic receptor (Conn et al., 2009). The use of such drugs in human cognitive paradigms has emphasised the role of the muscarinic M1 receptor in maintaining cognitive functioning (Nathan et al., 2013). This hypothesis is supported by preclinical studies showing muscarinic M1 receptor agonists are effective at modulating behavioural paradigms that require hippocampal engagement (Bradley et al., 2010, Vanover et al., 2008). Given the growing interest in targeting the muscarinic M1 receptor to try and modulate behaviours under the control of the hippocampus (Uslaner et al., 2013), we decided to determine the distribution of the muscarinic M1 receptor in the human hippocampus.
There have been studies on the muscarinic M1 receptor in the human CNS but these results must be treated with caution as some of the anti-muscarinic receptor antibodies used may lack specificity (Jositsch et al., 2009). Hence we began our study by obtaining data to support the specificity of the antibody to be used for our studies on the muscarinic M1 receptor.
Section snippets
Antibody Validation
Although the focus of our study was the human hippocampus, all validation experiments were completed using cortex because muscarinic M1 receptors are expressed at relatively high levels in that CNS region (Scarr et al., 2013). Frozen (Dean et al., 1999) and fixed tissue was used for these experiments.
Immunohistochemistry Human Hippocampus
Brain tissue was collected, following consent from the nearest next of kin, with the approval of the Ethics Committee of the Victorian Institute of Forensic Medicine. The tissue was sourced from
Antibody validation
Western blot analysis using the anti-muscarinic M1 receptor antibody showed immunogenic binding to a 62 kDa protein in cortical homogenates from human, wild type and Chrm4−/− mice; this protein was not detected in the cortex of Chrm1−/− mice (Fig. 1A). Using fixed tissue sections, the same antibody positively stained cells in cortex from wild type mice (Fig. 1B) and humans (Fig. 1D) but not in cortex from Chrm1−/− mice (Fig. 1C). Furthermore, as would be expected for a cellular receptor, the
Discussion
Given concerns about the specify of some muscarinic receptor antibodies for their target proteins (Jositsch et al., 2009) we have shown that the anti-muscarinic M1 receptor antibody used in this study does not show specific binding to any protein in cortex from mice lacking the Chrm1 gene. By contrast the antibody bound to a 62 KDa protein in cortical tissue from wild type mice, mice that do not express Chrm4 and human cortex. Moreover, the molecular weight of the antigenic protein for our
Acknowledgements
The authors thank Geoff Pavey for his curation of the human post-mortem tissue and Doris Tomas for her technical assistance. Tissues were received from the Victorian Brain Bank Network, supported by The Florey Institute of Neuroscience and Mental Health, The Alfred and the Victorian Forensic Institute of Medicine and funded in part by Australia’s National Health & Medical Research Council, Parkinson’s Victoria and MND Victoria. This study was also supported by Operational Infrastructure Support
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