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The distribution of muscarinic M1 receptors in the human hippocampus
Accepted Manuscript Title: The distribution of muscarinic M1 receptors in the human hippocampus Author: Elizabeth Scarr Myoung Suk Seo Timothy Douglas Aumann Gursharan Chana Ian Paul Everall Brian Dean PII: DOI: Reference:
S0891-0618(16)30061-8 http://dx.doi.org/doi:10.1016/j.jchemneu.2016.07.006 CHENEU 1422
To appear in: Received date: Revised date: Accepted date:
15-4-2016 13-7-2016 15-7-2016
Please cite this article as: Scarr, Elizabeth, Seo, Myoung Suk, Aumann, Timothy Douglas, Chana, Gursharan, Everall, Ian Paul, Dean, Brian, The distribution of muscarinic M1 receptors in the human hippocampus.Journal of Chemical Neuroanatomy http://dx.doi.org/10.1016/j.jchemneu.2016.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The distribution of muscarinic M1 receptors in the human hippocampus
Elizabeth Scarr,1,2 Myoung Suk Seo,1 Timothy Douglas Aumann,3 Gursharan Chana,4 Ian Paul Everall,2,5 and Brian Dean1
1
The Molecular Psychiatry Laboratory, The Florey Institute of Neuroscience and Mental Health,
The University of Melbourne, Victoria, Australia, 2
The Psychiatric Neuropathology Laboratory, the Department of Psychiatry, The University of
Melbourne, Victoria, Australia, 3
The Adult Neurogenesis Laboratory, The Florey Institute of Neuroscience and Mental Health, The
University of Melbourne, Victoria, Australia, 4
The Integrative Biological Psychiatry Laboratory, Centre for Neural Engineering, The University
of Melbourne, Parkville, Victoria, Australia, 5
The Department of Psychiatry, the North West Mental Health, Royal Melbourne Hospital,
Parkville, Victoria, Australia.
Corresponding Author: Professor Brian Dean, Head of the Biological Psychiatry and Mental Health Division and the Molecular Psychiatry, The Florey Institute for Neuroscience and Mental Health, 30 Royal Parade, Parkville, Victoria 3052, Australia. Ph: +61 3 8344 3786, Fax: +61 3 9348 1707, email: [email protected]
Grant Sponsor: NHMRC; Grant numbers: APP1045619, APP1002240 & APP1037234 Grant Sponsor: ARC; Grant numbers: FT100100689 & DP110100086
Keywords: immunohistochemistry, acetylcholine, post-mortem, neurons, glutamate 1
Highlights
A muscarinic M1 receptor antibody is validated for specificity using Western blotting and immunohistochemistry using CNS from wild type and muscarinic M1 receptor knockout mice.
This antibody is used to visualise the distribution of muscarinic M1 receptors in the human 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.
Immunostaining suggested some muscarinic M1 receptors were not localised to the cellular membrane. These receptors may be the muscarinic M1 receptor reserve or internalised receptors being degraded.
2
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.
3
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.
4
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.
5
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.
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Materials and Methods:
Tissue Collection and Processing
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 the Victorian Brain Bank at the Florey Institute of Neuroscience and Mental Health. Details pertaining to the ten cases from whom tissue was collected and who had no history of psychiatric or neurological disorders are in Table 1.
Tissue for immunohistochemistry required the removal of the whole brain from the cranium, the brains were hemisected with the right hemisphere being fixed in 37% formaldehyde for at least two weeks before being processed for neuropathology using a standardised process. The slices (1cm thickness) from the neuropathology cut were then stored in 10% neutral buffered formalin. The hippocampal blocks for this study were taken at the level of the lateral geniculate nucleus. After removal, the blocks were placed in phosphate buffered saline (PBS). The blocks were serially sectioned at 50 m on a vibratome and stored in PBS containing 0.5% sodium azide at 4°C. 7
Immunohistochemistry Mouse CNS
With permission of the Animal Ethics Committee of the Florey Institute for Neuroscience and Mental Health, CNS was removed from muscarinic M1 (Chrm1-\-) and M4 (Chrm4-\-) receptor knock out mice and wild type (C57BL/6-NTAC) mice (n = 5 per group), fixed as for human tissue, embedded in paraffin wax and serially sectioned at 7 um on a rotary microtome before being stored at room temperature. The sections used for this study were taken at the level of Bregma 1.18mm (Franklin and Paxinos, 2008).
Antibody Validation
The polyclonal anti-muscarinic M1 receptor antibody used in this study (mAChR-M1-Rb; batch Af340) was raised against amino acids 247-345 of the mouse muscarinic M1 receptor (NM_007698) (Narushima et al.,2007) and was purchased from Frontier Institute Co. Ltd. Hokkaido, Japan. The specificity of the antibody was explored by i) using Western blot and fresh frozen samples of cortex from Chrm1-/-, Chrm4-/- and wild type mice plus a sample of human cortex and ii) immunohistochemistry using fixed brain tissue from Chrm1-\- and wild type mice plus human cortex.
Western blotting
CNS tissue (~50mg) from Chrm1-\-, Chrm4-\-, wild type mice and a human case were homogenised (10% w/v) into 10 mM Tris-HCl (pH 7.0) containing 320 mM Sucrose, 1 mM EDTA and 20 mM KCl. Homogenates were centrifuged at 1000 x g for 10 minutes at 4°C and the supernatant recovered. Protein concentrations were determined using the Bio-Rad
8
modified Lowry protein assay adapted for the microplate. The homogenates were diluted in reducing buffer (0.125 M Tris-HCl pH 6.8, 20% glycerol, 100 mM dithiothreitol, 4% SDS, 0.0025% bromophenol blue) to give a final concentration of 1.5mg/ml. Samples were denatured by heating at 55°C for 30 minutes, briefly centrifuged and 20l of each sample loaded onto a polyacrylamide gel (4% loading, 10% stacking gel) alongside BioRad broad range molecular weight standards and separated using a constant voltage (150V).
The
proteins were then transferred to nitrocellulose membranes overnight in Towbin transfer buffer using a constant current (40mA). Loading and uniform transfer were confirmed using 0.1% Ponceau S in 3% trichloroacetic acid. The nitrocellulose was blocked for 1 hour in antibody diluent (5% non-fat milk powder (NFMP) in Tris buffered saline containing 0.1% Tween-20; TTBS) then incubated with rabbit anti-CHRM1 antibody (Frontier Institute Co. Ltd; mAChR-M1-Rb; batch Af340; 1/100 in antibody diluent) overnight at 4°C. This was followed by incubation with goat anti-rabbit antibody conjugated to horseradish peroxidise (1/2000 in TTBS: DAKO; P0448; batch 200110775) for an hour at 21°C. The antigenic reaction was visualised using enhanced chemiluminescence (ECL) and imaged using a Kodak Image Station 440CF. The sum intensity of the antigenic bands was measured using Kodak 1D software and the molecular weights calculated with reference to the standards.
Immunohistochemistry
Immunohistochemistry was performed using the Vectastain ABC peroxidase kit (Rabbit IgG, PK4001). Free-floating sections were removed from storage solution and washed in PBS before being incubated in 1% hydrogen peroxide in methanol to quench endogenous peroxidase activity. The sections were washed in deionised water and PBS, microwaved in citrate buffer (pH 6.0; Abcam; Cat# 64236; Lot# GR80382-1) to retrieve antigens and
9
washed in PBS before being blocked in antibody diluent (1% normal goat serum in 5% NFMP in PBS containing 0.1% Triton x-100) for two hours at 21°C. Tissue sections were then incubated with the primary antibody (mAChR-M1-Rb; batch Af340; 1/67 in antibody diluent) overnight at 4°C. Following washes in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG secondary antibody (Vectastain; 1/200 dilution in PBS containing 0.1% Triton x-100) for two hours at 21°C. After washes in PBS, the sections were incubated in PBS containing Avidin-DH (1/100) and biotinylated peroxidase H (1/100) for two hours at 21°C. Sections were washed in PBS before being incubated with 3, 3’diaminobenzidine (Vector ImmPACT™, SK4105; 1/33 in supplied diluent) for 90 seconds at 21°C, followed by washes in deionised water and PBS. Sections were mounted on slides and air-dried before being counterstained with haematoxylin (Vector® Hematoxylin Nuclear Counterstain (Gill’s Formula); H-3401; Lot #Z0916) for 210 seconds at 21°C, washed and then dehydrated through graded alcohols, cleared in histolene and permanently mounted in DPX.
To aid with the identification for hippocampal regions, 1 section from each subject was stained using cresyl violet. Following incubation with 0.1% cresyl violet acetate in deionised water for 20 minutes at room temperature, sections were washed and dehydrated through gradient alcohols, cleared in histolene and permanently mounted in DPX.
Sections were imaged using an Eclipse 80i microscope (Nikon, Japan), linked to a computer running Image Pro-Plus 7.0 (MediaCybernetics, USA) following automatic white balance. The 1x image of the hippocampus was acquired with a Micro-Nikkor 55mm f/2/8 lens (Nikon, Japan), linked to a computer running Spot Software version 4.6 using automatic white balance. 10
RESULTS
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 (Figure 1A). Using fixed tissue sections, the same antibody positively stained cells in cortex from wild type mice (Figure 1B) and humans (Figure 1D) but not in cortex from Chrm1-/- mice (Figure1C). Furthermore, as would be expected for a cellular receptor, the immunoperoxidase staining was generally constrained to cell bodies and proximal dendrites (Figure 1B and D).
Human Hippocampus
In this study, we employed a cytoarchitectural nomenclature widely used for human hippocampal studies (Figure 2A) (Insausti and Amaral, 2011) with observations being consistent across all 10 cases. Muscarinic M1 receptor positive staining was present in the deep polymorphic layer (PM), less obvious in the granular (G) and sporadic in the molecular (M) layer of the dentate gyrus (Figure 2B). Within the cornu ammonis (CA), positive staining was almost exclusively limited to the pyramidal layer with positive staining being strongest in CA3, less intense in CA2 and sparsely distributed in CA1. Moderate levels of positive staining was present in the subiculum (SC, pyramidal layer), the presubiculum (PS, layer II) and cortical layers (III and V) of the parahippocampal gyrus (PHG).
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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 current antibody is essentially the same as that of the immunogenic protein we had identified as the muscarinic M1 receptor in human cortex using a different anti-muscarinic M1 receptor antibody (Dean et al., 2002) and as identified by others in human keratinocytes (Ndoye et al., 1998) and the rabbit retina (Strang et al., 2010). These data give strong support to the argument that the antibody used in this study shows specificity for the human and mammalian muscarinic M1 receptor.
Similar to our study, a previous study has reported muscarinic M1 receptors as being present on pyramidal neurons in all CA regions and, as in our study, being predominant in CA 3 and CA 2 (Shiozaki et al., 2001). However, this study also found muscarinic M1 receptors on granular neurons in the stratum granulosum of the dentate gyrus, non-pyramidal neurons within the polymorphic layer of the dentate gyrus, on cell bodies in the stratum pyramidale and apical dendrites in the stratum radiatum. Again, like our study, muscarinic M1 receptors were identified in the subiculum and parahippocampal gyrus. Thus this study found a wider cell-type distribution of muscarinic M1 receptors in the human hippocampus. Importantly, the antibody used in this study was shown to immunoprecipitate [3H]QNB, a pan-muscarinic receptor radioligand, bound to cloned human muscarinic M1 receptor but no Western blot of
12
human CNS has given to confirm the anti-muscarinic M1 receptor antibody was specific to a single immunogenic site (Shiozaki et al., 1999). Thus, it remains possible this antibody could bind to more than a single protein in human CNS and this could account for the more diverse binding reported when using this antibody in immunohistochemistry with human tissue.
Similar to the previous study using human tissue, our study shows more restricted distribution muscarinic M1 receptors the human hippocampus to what has been reported using a different antibody and tissue from non-human primates (Levey, 1996). Most striking is the difference in distribution in the dentate gyrus where the study on non-human primate reports high receptor levels in the granular and molecular layers. Our study does show muscarinic M1 receptor immunoreactivity in the granular layer but such immunoreactive appears more prevalent in the deep polymorphic layer of this hippocampal region. Our study also found that muscarinic M1 receptor positive cells were predominantly present in the pyramidal layers of CA3 and CA2, with less intense staining in CA1. This again contrasts with the nonhuman primate study which reports similar intensities of the receptor in the pyramidal layer and the lacunosum moleculare of all CA regions, with slightly higher levels in the stratum radiatum to polymorphic layers. These data, added to other data on the distribution of markers for cholinergic systems between humans and rhesus monkeys (Macaca mulatta) (Green and Mesulam, 1988), suggest there may have been some divergence in the structure and function of CNS cholinergic pathways between humans and primates.
We have reported on the distribution of muscarinic M1 receptor mRNA in human hippocampus (Scarr et al., 2007). The highest levels of muscarinic M1 receptor mRNA was in the granular layer of the dentate gyrus whereas the muscarinic M1 receptor protein is most prominent in the polymorphic layer, with lower levels in the molecular layer. Muscarinic M1
13
receptor mRNA was highest in the pyramidal layer of CA2 but present throughout CA1, CA2 and CA3. Notably, the muscarinic M1 receptor protein was limited to the pyramidal layer in CA1, CA2 and CA3 with levels of protein staining being moderate in the alveus, stratum oriens and pyramidal layer and low in the lacunosum moleculare and stratum radiatum of CA1. The presence of mRNA in human pyramidal cells matches reports of the presents of muscarinic M1 receptor mRNA in pyramidal cells in hippocampi from both human and nonhuman primates (Levey, 1996).
A close examination of the distribution of muscarinic M1 receptors in various regions of the hippocampus suggests localisation of receptors to the cell membrane and in the cytosol. In the mouse hippocampus it has been reported that there is a high level of muscarinic M1 receptor reserve (Felder et al., 2001). It is therefore possible that the cellular distribution of muscarinic M1 receptors observed in our study could be reflecting a reserve of nonmembrane bound receptors in human hippocampus.
Alternatively, the degradation of
muscarinic M1 receptors involves internalisation by endocytosis (Davis et al., 2009) and the apparent cytosolic distribution of staining we observe may represent muscarinic M1 receptors being removed from the cellular membrane and being degraded. Further experiments using chemical derivatisation (Felder et al., 2001) plus confocal and / or electron microscopy would be warranted to better understand the cause of the apparent cytosolic distribution of muscarinic M1 receptors in the human hippocampus.
One rationale for the timeliness of our study was that muscarinic M1 receptor specific drugs have been synthesised (Conn et al., 2009) and are now entering clinical trials and knowing where their target receptor was located would help define their mechanism of action. Hence, the localisation of muscarinic M1 receptors on pyramidal neurons in the human hippocampus
14
means they are ideally located to modulate acetylcholine mediation of glutamatergic neurotransmission (Alcantara et al., 2001; Bradshaw et al., 1987). This notion is reinforced by the finding that cortical glutamatergic neurons from mice lacking the muscarinic m1 receptor did not respond normally to either tonic or phasic acetylcholine (Gulledge et al., 2009). These animals also had deficits in cue detection paradigms indicating there is a physiological effect associated with the impaired cholinergic modulation of glutamatergic transmission.
Moving beyond neurochemical associations, activating muscarinic receptors has been shown to generate gamma oscillations (Palhalmi et al., 2004), which are thought to be important for focussed attention and spike timing-dependant synaptic plasticity, requiring a balance of excitatory and inhibitory activity. It has been suggested this activation involves muscarinic M1 receptors on pyramidal cells because it is non-existent in Chrm1-/- mice (Fisahn et al., 2002) and tonic and phasic cholinergic responses in CA1and 3 pyramidal cells (Dasari and Gulledge, 2011) is severely curtailed in these mice. This, combined with the report that Chrm1-/- mice show a reduced long term potentiation in response to hippocampal theta bursts (Anagnostaras et al., 2003), suggests muscarinic M1 receptors play a vital role in the cellular processes underlying learning and memory. If this proves to be a mechanism of action of drugs such as positive allosteric modulators of the muscarinic M1 receptors then such drugs should be useful in treating disorders where learning and memory are affected.
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Acknowledgements: The authors thank Geoff 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 (OIS) from the Victorian State Government.
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Figure Legends:
Figure 1A: An image, with molecular weight markers shown on the left and lane numbers above, of Western blot where all lanes contain 30g of total protein and visualised using an anti-muscarinic M1 receptor antibody with chemiluminescence. Lane 1 = human cortex, lane 2 = wild type mouse cortex, lane 3 = Chrm1-/- and lane 4 = Chrm4-/- cortex. The molecular weight of the single band seen in human cortex and present in all samples except that from the m1-/- mouse is shown on the right. 1B - D: The staining of muscarinic M1 receptors using anti-muscarinic M1 receptor antibody showing positively stained cells in cortex from wild type mice (B) and a human (D), but not Chrm1-/- (C). Magnification at x1 in the upper panels and x 40x in the lower panels; the scale bar indicates 1000 m on 1X and 100 m on 40X.
Figure 2: Muscarinic M1 receptors in human hippocampus at x1 magnification showing the whole hippocampal formation (A) and at x 40 magnification (B) showing the deep polymorphic (PM) granular (G) and molecular (M) layers of the dentate gyrus (DG), the pyramidal layers of CA3, CA2 and CA1, the subicular “clouds” (SC), the pre-subiculum (PS) and the parahippocampal gyrus (PHG). Tissue staining in CA2 at x 40 magnification in the absence of primary antibody (C) is also shown. The scale bar indicates 1.0 cm on 1X and 100 m on 40X.
17
Table 1: Demographic and tissue collection information for the subjects whose hippocampal tissue was used in this study. Subject ID
Age
Sex
Cause of Death
(Years)
Post-mortem Interval
Brain pH
(hours)
C1
36
M
Crush accident
42
6.46
C2
42
M
Cardiomegaly
63
6.34
C3
21
F
Myocarditis
58
6.03
C4
21
M
Acute epiglottitis
40
5.82
C5
25
M
Electrocution
24
6.42
C6
75
M
Cardiogenic shock
69.4
6.19
C7
52
M
Cardiomegaly
33.75
6.52
C8
66
F
Infrarenal atherosclerosis
49.25
6.44
C9
52
M
Ischaemic heart disease
50
6.78
C10
66
M
Coronary artery atheroma
71.75
6.47
18
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Strang, C.E., Renna, J.M., Amthor, F.R., Keyser, K.T., 2010. Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties. Invest. Ophthalmol. Vis. Sci. 51, 2778-2789.
Uslaner, J.M., Eddins, D., Puri, V., Cannon, C.E., Sutcliffe, J., Chew, C.S., Pearson, M., Vivian, J.A., Chang, R.K., Ray, W.J., Kuduk, S.D., Wittmann, M., 2013. The muscarinic M1 receptor positive allosteric modulator PQCA improves cognitive measures in rat, cynomolgus macaque, and rhesus macaque. Psychopharmacol. (Berlin) 225, 21-30.
Vanover, K.E., Veinbergs, I., Davis, R.E., 2008. Antipsychotic-like behavioral effects and cognitive enhancement by a potent and selective muscarinic M-sub-1 receptor agonist, AC260584. Behav. Neurosci. 122, 570-575.
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S0891-0618(16)30061-8 http://dx.doi.org/doi:10.1016/j.jchemneu.2016.07.006 CHENEU 1422
To appear in: Received date: Revised date: Accepted date:
15-4-2016 13-7-2016 15-7-2016
Please cite this article as: Scarr, Elizabeth, Seo, Myoung Suk, Aumann, Timothy Douglas, Chana, Gursharan, Everall, Ian Paul, Dean, Brian, The distribution of muscarinic M1 receptors in the human hippocampus.Journal of Chemical Neuroanatomy http://dx.doi.org/10.1016/j.jchemneu.2016.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The distribution of muscarinic M1 receptors in the human hippocampus
Elizabeth Scarr,1,2 Myoung Suk Seo,1 Timothy Douglas Aumann,3 Gursharan Chana,4 Ian Paul Everall,2,5 and Brian Dean1
1
The Molecular Psychiatry Laboratory, The Florey Institute of Neuroscience and Mental Health,
The University of Melbourne, Victoria, Australia, 2
The Psychiatric Neuropathology Laboratory, the Department of Psychiatry, The University of
Melbourne, Victoria, Australia, 3
The Adult Neurogenesis Laboratory, The Florey Institute of Neuroscience and Mental Health, The
University of Melbourne, Victoria, Australia, 4
The Integrative Biological Psychiatry Laboratory, Centre for Neural Engineering, The University
of Melbourne, Parkville, Victoria, Australia, 5
The Department of Psychiatry, the North West Mental Health, Royal Melbourne Hospital,
Parkville, Victoria, Australia.
Corresponding Author: Professor Brian Dean, Head of the Biological Psychiatry and Mental Health Division and the Molecular Psychiatry, The Florey Institute for Neuroscience and Mental Health, 30 Royal Parade, Parkville, Victoria 3052, Australia. Ph: +61 3 8344 3786, Fax: +61 3 9348 1707, email: [email protected]
Grant Sponsor: NHMRC; Grant numbers: APP1045619, APP1002240 & APP1037234 Grant Sponsor: ARC; Grant numbers: FT100100689 & DP110100086
Keywords: immunohistochemistry, acetylcholine, post-mortem, neurons, glutamate 1
Highlights
A muscarinic M1 receptor antibody is validated for specificity using Western blotting and immunohistochemistry using CNS from wild type and muscarinic M1 receptor knockout mice.
This antibody is used to visualise the distribution of muscarinic M1 receptors in the human 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.
Immunostaining suggested some muscarinic M1 receptors were not localised to the cellular membrane. These receptors may be the muscarinic M1 receptor reserve or internalised receptors being degraded.
2
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.
3
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.
4
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.
5
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.
6
Materials and Methods:
Tissue Collection and Processing
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 the Victorian Brain Bank at the Florey Institute of Neuroscience and Mental Health. Details pertaining to the ten cases from whom tissue was collected and who had no history of psychiatric or neurological disorders are in Table 1.
Tissue for immunohistochemistry required the removal of the whole brain from the cranium, the brains were hemisected with the right hemisphere being fixed in 37% formaldehyde for at least two weeks before being processed for neuropathology using a standardised process. The slices (1cm thickness) from the neuropathology cut were then stored in 10% neutral buffered formalin. The hippocampal blocks for this study were taken at the level of the lateral geniculate nucleus. After removal, the blocks were placed in phosphate buffered saline (PBS). The blocks were serially sectioned at 50 m on a vibratome and stored in PBS containing 0.5% sodium azide at 4°C. 7
Immunohistochemistry Mouse CNS
With permission of the Animal Ethics Committee of the Florey Institute for Neuroscience and Mental Health, CNS was removed from muscarinic M1 (Chrm1-\-) and M4 (Chrm4-\-) receptor knock out mice and wild type (C57BL/6-NTAC) mice (n = 5 per group), fixed as for human tissue, embedded in paraffin wax and serially sectioned at 7 um on a rotary microtome before being stored at room temperature. The sections used for this study were taken at the level of Bregma 1.18mm (Franklin and Paxinos, 2008).
Antibody Validation
The polyclonal anti-muscarinic M1 receptor antibody used in this study (mAChR-M1-Rb; batch Af340) was raised against amino acids 247-345 of the mouse muscarinic M1 receptor (NM_007698) (Narushima et al.,2007) and was purchased from Frontier Institute Co. Ltd. Hokkaido, Japan. The specificity of the antibody was explored by i) using Western blot and fresh frozen samples of cortex from Chrm1-/-, Chrm4-/- and wild type mice plus a sample of human cortex and ii) immunohistochemistry using fixed brain tissue from Chrm1-\- and wild type mice plus human cortex.
Western blotting
CNS tissue (~50mg) from Chrm1-\-, Chrm4-\-, wild type mice and a human case were homogenised (10% w/v) into 10 mM Tris-HCl (pH 7.0) containing 320 mM Sucrose, 1 mM EDTA and 20 mM KCl. Homogenates were centrifuged at 1000 x g for 10 minutes at 4°C and the supernatant recovered. Protein concentrations were determined using the Bio-Rad
8
modified Lowry protein assay adapted for the microplate. The homogenates were diluted in reducing buffer (0.125 M Tris-HCl pH 6.8, 20% glycerol, 100 mM dithiothreitol, 4% SDS, 0.0025% bromophenol blue) to give a final concentration of 1.5mg/ml. Samples were denatured by heating at 55°C for 30 minutes, briefly centrifuged and 20l of each sample loaded onto a polyacrylamide gel (4% loading, 10% stacking gel) alongside BioRad broad range molecular weight standards and separated using a constant voltage (150V).
The
proteins were then transferred to nitrocellulose membranes overnight in Towbin transfer buffer using a constant current (40mA). Loading and uniform transfer were confirmed using 0.1% Ponceau S in 3% trichloroacetic acid. The nitrocellulose was blocked for 1 hour in antibody diluent (5% non-fat milk powder (NFMP) in Tris buffered saline containing 0.1% Tween-20; TTBS) then incubated with rabbit anti-CHRM1 antibody (Frontier Institute Co. Ltd; mAChR-M1-Rb; batch Af340; 1/100 in antibody diluent) overnight at 4°C. This was followed by incubation with goat anti-rabbit antibody conjugated to horseradish peroxidise (1/2000 in TTBS: DAKO; P0448; batch 200110775) for an hour at 21°C. The antigenic reaction was visualised using enhanced chemiluminescence (ECL) and imaged using a Kodak Image Station 440CF. The sum intensity of the antigenic bands was measured using Kodak 1D software and the molecular weights calculated with reference to the standards.
Immunohistochemistry
Immunohistochemistry was performed using the Vectastain ABC peroxidase kit (Rabbit IgG, PK4001). Free-floating sections were removed from storage solution and washed in PBS before being incubated in 1% hydrogen peroxide in methanol to quench endogenous peroxidase activity. The sections were washed in deionised water and PBS, microwaved in citrate buffer (pH 6.0; Abcam; Cat# 64236; Lot# GR80382-1) to retrieve antigens and
9
washed in PBS before being blocked in antibody diluent (1% normal goat serum in 5% NFMP in PBS containing 0.1% Triton x-100) for two hours at 21°C. Tissue sections were then incubated with the primary antibody (mAChR-M1-Rb; batch Af340; 1/67 in antibody diluent) overnight at 4°C. Following washes in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG secondary antibody (Vectastain; 1/200 dilution in PBS containing 0.1% Triton x-100) for two hours at 21°C. After washes in PBS, the sections were incubated in PBS containing Avidin-DH (1/100) and biotinylated peroxidase H (1/100) for two hours at 21°C. Sections were washed in PBS before being incubated with 3, 3’diaminobenzidine (Vector ImmPACT™, SK4105; 1/33 in supplied diluent) for 90 seconds at 21°C, followed by washes in deionised water and PBS. Sections were mounted on slides and air-dried before being counterstained with haematoxylin (Vector® Hematoxylin Nuclear Counterstain (Gill’s Formula); H-3401; Lot #Z0916) for 210 seconds at 21°C, washed and then dehydrated through graded alcohols, cleared in histolene and permanently mounted in DPX.
To aid with the identification for hippocampal regions, 1 section from each subject was stained using cresyl violet. Following incubation with 0.1% cresyl violet acetate in deionised water for 20 minutes at room temperature, sections were washed and dehydrated through gradient alcohols, cleared in histolene and permanently mounted in DPX.
Sections were imaged using an Eclipse 80i microscope (Nikon, Japan), linked to a computer running Image Pro-Plus 7.0 (MediaCybernetics, USA) following automatic white balance. The 1x image of the hippocampus was acquired with a Micro-Nikkor 55mm f/2/8 lens (Nikon, Japan), linked to a computer running Spot Software version 4.6 using automatic white balance. 10
RESULTS
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 (Figure 1A). Using fixed tissue sections, the same antibody positively stained cells in cortex from wild type mice (Figure 1B) and humans (Figure 1D) but not in cortex from Chrm1-/- mice (Figure1C). Furthermore, as would be expected for a cellular receptor, the immunoperoxidase staining was generally constrained to cell bodies and proximal dendrites (Figure 1B and D).
Human Hippocampus
In this study, we employed a cytoarchitectural nomenclature widely used for human hippocampal studies (Figure 2A) (Insausti and Amaral, 2011) with observations being consistent across all 10 cases. Muscarinic M1 receptor positive staining was present in the deep polymorphic layer (PM), less obvious in the granular (G) and sporadic in the molecular (M) layer of the dentate gyrus (Figure 2B). Within the cornu ammonis (CA), positive staining was almost exclusively limited to the pyramidal layer with positive staining being strongest in CA3, less intense in CA2 and sparsely distributed in CA1. Moderate levels of positive staining was present in the subiculum (SC, pyramidal layer), the presubiculum (PS, layer II) and cortical layers (III and V) of the parahippocampal gyrus (PHG).
11
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 current antibody is essentially the same as that of the immunogenic protein we had identified as the muscarinic M1 receptor in human cortex using a different anti-muscarinic M1 receptor antibody (Dean et al., 2002) and as identified by others in human keratinocytes (Ndoye et al., 1998) and the rabbit retina (Strang et al., 2010). These data give strong support to the argument that the antibody used in this study shows specificity for the human and mammalian muscarinic M1 receptor.
Similar to our study, a previous study has reported muscarinic M1 receptors as being present on pyramidal neurons in all CA regions and, as in our study, being predominant in CA 3 and CA 2 (Shiozaki et al., 2001). However, this study also found muscarinic M1 receptors on granular neurons in the stratum granulosum of the dentate gyrus, non-pyramidal neurons within the polymorphic layer of the dentate gyrus, on cell bodies in the stratum pyramidale and apical dendrites in the stratum radiatum. Again, like our study, muscarinic M1 receptors were identified in the subiculum and parahippocampal gyrus. Thus this study found a wider cell-type distribution of muscarinic M1 receptors in the human hippocampus. Importantly, the antibody used in this study was shown to immunoprecipitate [3H]QNB, a pan-muscarinic receptor radioligand, bound to cloned human muscarinic M1 receptor but no Western blot of
12
human CNS has given to confirm the anti-muscarinic M1 receptor antibody was specific to a single immunogenic site (Shiozaki et al., 1999). Thus, it remains possible this antibody could bind to more than a single protein in human CNS and this could account for the more diverse binding reported when using this antibody in immunohistochemistry with human tissue.
Similar to the previous study using human tissue, our study shows more restricted distribution muscarinic M1 receptors the human hippocampus to what has been reported using a different antibody and tissue from non-human primates (Levey, 1996). Most striking is the difference in distribution in the dentate gyrus where the study on non-human primate reports high receptor levels in the granular and molecular layers. Our study does show muscarinic M1 receptor immunoreactivity in the granular layer but such immunoreactive appears more prevalent in the deep polymorphic layer of this hippocampal region. Our study also found that muscarinic M1 receptor positive cells were predominantly present in the pyramidal layers of CA3 and CA2, with less intense staining in CA1. This again contrasts with the nonhuman primate study which reports similar intensities of the receptor in the pyramidal layer and the lacunosum moleculare of all CA regions, with slightly higher levels in the stratum radiatum to polymorphic layers. These data, added to other data on the distribution of markers for cholinergic systems between humans and rhesus monkeys (Macaca mulatta) (Green and Mesulam, 1988), suggest there may have been some divergence in the structure and function of CNS cholinergic pathways between humans and primates.
We have reported on the distribution of muscarinic M1 receptor mRNA in human hippocampus (Scarr et al., 2007). The highest levels of muscarinic M1 receptor mRNA was in the granular layer of the dentate gyrus whereas the muscarinic M1 receptor protein is most prominent in the polymorphic layer, with lower levels in the molecular layer. Muscarinic M1
13
receptor mRNA was highest in the pyramidal layer of CA2 but present throughout CA1, CA2 and CA3. Notably, the muscarinic M1 receptor protein was limited to the pyramidal layer in CA1, CA2 and CA3 with levels of protein staining being moderate in the alveus, stratum oriens and pyramidal layer and low in the lacunosum moleculare and stratum radiatum of CA1. The presence of mRNA in human pyramidal cells matches reports of the presents of muscarinic M1 receptor mRNA in pyramidal cells in hippocampi from both human and nonhuman primates (Levey, 1996).
A close examination of the distribution of muscarinic M1 receptors in various regions of the hippocampus suggests localisation of receptors to the cell membrane and in the cytosol. In the mouse hippocampus it has been reported that there is a high level of muscarinic M1 receptor reserve (Felder et al., 2001). It is therefore possible that the cellular distribution of muscarinic M1 receptors observed in our study could be reflecting a reserve of nonmembrane bound receptors in human hippocampus.
Alternatively, the degradation of
muscarinic M1 receptors involves internalisation by endocytosis (Davis et al., 2009) and the apparent cytosolic distribution of staining we observe may represent muscarinic M1 receptors being removed from the cellular membrane and being degraded. Further experiments using chemical derivatisation (Felder et al., 2001) plus confocal and / or electron microscopy would be warranted to better understand the cause of the apparent cytosolic distribution of muscarinic M1 receptors in the human hippocampus.
One rationale for the timeliness of our study was that muscarinic M1 receptor specific drugs have been synthesised (Conn et al., 2009) and are now entering clinical trials and knowing where their target receptor was located would help define their mechanism of action. Hence, the localisation of muscarinic M1 receptors on pyramidal neurons in the human hippocampus
14
means they are ideally located to modulate acetylcholine mediation of glutamatergic neurotransmission (Alcantara et al., 2001; Bradshaw et al., 1987). This notion is reinforced by the finding that cortical glutamatergic neurons from mice lacking the muscarinic m1 receptor did not respond normally to either tonic or phasic acetylcholine (Gulledge et al., 2009). These animals also had deficits in cue detection paradigms indicating there is a physiological effect associated with the impaired cholinergic modulation of glutamatergic transmission.
Moving beyond neurochemical associations, activating muscarinic receptors has been shown to generate gamma oscillations (Palhalmi et al., 2004), which are thought to be important for focussed attention and spike timing-dependant synaptic plasticity, requiring a balance of excitatory and inhibitory activity. It has been suggested this activation involves muscarinic M1 receptors on pyramidal cells because it is non-existent in Chrm1-/- mice (Fisahn et al., 2002) and tonic and phasic cholinergic responses in CA1and 3 pyramidal cells (Dasari and Gulledge, 2011) is severely curtailed in these mice. This, combined with the report that Chrm1-/- mice show a reduced long term potentiation in response to hippocampal theta bursts (Anagnostaras et al., 2003), suggests muscarinic M1 receptors play a vital role in the cellular processes underlying learning and memory. If this proves to be a mechanism of action of drugs such as positive allosteric modulators of the muscarinic M1 receptors then such drugs should be useful in treating disorders where learning and memory are affected.
15
Acknowledgements: The authors thank Geoff 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 (OIS) from the Victorian State Government.
16
Figure Legends:
Figure 1A: An image, with molecular weight markers shown on the left and lane numbers above, of Western blot where all lanes contain 30g of total protein and visualised using an anti-muscarinic M1 receptor antibody with chemiluminescence. Lane 1 = human cortex, lane 2 = wild type mouse cortex, lane 3 = Chrm1-/- and lane 4 = Chrm4-/- cortex. The molecular weight of the single band seen in human cortex and present in all samples except that from the m1-/- mouse is shown on the right. 1B - D: The staining of muscarinic M1 receptors using anti-muscarinic M1 receptor antibody showing positively stained cells in cortex from wild type mice (B) and a human (D), but not Chrm1-/- (C). Magnification at x1 in the upper panels and x 40x in the lower panels; the scale bar indicates 1000 m on 1X and 100 m on 40X.
Figure 2: Muscarinic M1 receptors in human hippocampus at x1 magnification showing the whole hippocampal formation (A) and at x 40 magnification (B) showing the deep polymorphic (PM) granular (G) and molecular (M) layers of the dentate gyrus (DG), the pyramidal layers of CA3, CA2 and CA1, the subicular “clouds” (SC), the pre-subiculum (PS) and the parahippocampal gyrus (PHG). Tissue staining in CA2 at x 40 magnification in the absence of primary antibody (C) is also shown. The scale bar indicates 1.0 cm on 1X and 100 m on 40X.
17
Table 1: Demographic and tissue collection information for the subjects whose hippocampal tissue was used in this study. Subject ID
Age
Sex
Cause of Death
(Years)
Post-mortem Interval
Brain pH
(hours)
C1
36
M
Crush accident
42
6.46
C2
42
M
Cardiomegaly
63
6.34
C3
21
F
Myocarditis
58
6.03
C4
21
M
Acute epiglottitis
40
5.82
C5
25
M
Electrocution
24
6.42
C6
75
M
Cardiogenic shock
69.4
6.19
C7
52
M
Cardiomegaly
33.75
6.52
C8
66
F
Infrarenal atherosclerosis
49.25
6.44
C9
52
M
Ischaemic heart disease
50
6.78
C10
66
M
Coronary artery atheroma
71.75
6.47
18
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