Elsevier

Journal of Chemical Neuroanatomy

Volume 77, November 2016, Pages 187-192
Journal of Chemical Neuroanatomy

The distribution of muscarinic M1 receptors in the human hippocampus

https://doi.org/10.1016/j.jchemneu.2016.07.006Get rights and content

Highlights

Abstract

The muscarinic M1 receptor plays a significant role in cognition, probably by modulating information processing in key regions such as the hippocampus. To understand how the muscarinic M1 receptor achieves these functions in the hippocampus, it is critical to know the distribution of the receptor within this complex brain region. To date, there are limited data on the distribution of muscarinic M1 receptors in the human hippocampus which may also be confounded because some anti-muscarinic receptor antibodies have been shown to lack specificity.

Initially, using Western blotting and immunohistochemistry, we showed the anti-muscarinic M1 receptor antibody to be used in our study bound to a single 62 kDa protein that was absent in mice lacking the muscarinic M1 receptor gene. Then, using immunohistochemistry, we determined the distribution of muscarinic M1 receptors in human hippocampus from 10 subjects with no discernible history of a neurological or psychiatric disorder.

Our data shows the muscarinic M1 receptor to be predominantly on pyramidal cells in the hippocampus. Muscarinic M1 receptor positive cells were most apparent in the deep polymorphic layer of the dentate gyrus, the pyramidal cell layer of cornu ammonis region 3, the cellular layers of the subiculum, layer II of the presubiculum and layer III and V of the parahippocampal gyrus. Positive cells were less numerous and less intensely stained in the pyramidal layer of cornu ammonis region 2 and were sparse in the molecular layer of the dentate gyrus as well as cornu ammonis region 1. Although immunoreactivity was present in the granular layer of the dentate gyrus, it was difficult to identity individual immunopositive cells, possibly due to the density of cells.

This distribution of the muscarinic M1 receptors in human hippocampus, and its localisation on glutamatergic cells, would suggest the receptor has a significant role in modulating excitatory hippocampal neurotransmission.

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

References (33)

  • E. Scarr et al.

    Altered hippocampal muscarinic m4, but not m1, receptor expression from subjects with schizophrenia

    Biol. Psychiatr.

    (2007)
  • K. Shiozaki et al.

    Distribution of m1 muscarinic acetylcholine receptors in the hippocampus of patients with Alzheimer's disease and dementia with Lewy bodies-an immunohistochemical study

    J. Neurol. Sci.

    (2001)
  • A.A. Alcantara et al.

    Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways

    J. Comp. Neurol.

    (2001)
  • S.G. Anagnostaras et al.

    Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice

    Nat. Neurosci.

    (2003)
  • S. Dasari et al.

    M1 and M4 receptors modulate hippocampal pyramidal neurons

    J. Neurophysiol.

    (2011)
  • C.N. Davis et al.

    Differential regulation of muscarinic M1 receptors by orthosteric and allosteric ligands

    BMC Pharmacol.

    (2009)
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