TYPE Review
PUBLISHED

22 February 2023

DOI 10.3389/fncel.2023.1124333

OPEN ACCESSEDITED BY

Eleonora Napoli,
The University of California, Davis,
United States
REVIEWED BY

Hugo Geerts,
Certara UK Limited, United Kingdom
John Cookson,
East London NHS Foundation Trust,
United Kingdom

A growing understanding of therole of muscarinic receptors in
the molecular pathology andtreatment of schizophrenia
Brian Dean1*, Geor Bakker2 , Hiroki R. Ueda3,4 , Andrew B. Tobin5 ,Alastair Brown2 and Richard A. A. Kanaan6
1*CORRESPONDENCE

Brian Dean
brian.dean@florey.edu.au
SPECIALTY SECTION

This article was submitted to
Cellular Neuropathology,
a section of the journal
Frontiers in Cellular Neuroscience
RECEIVED 15 December 2022
ACCEPTED 06 February 2023
PUBLISHED 22 February 2023
CITATION

Dean B, Bakker G, Ueda HR, Tobin AB,
Brown A and Kanaan RAA (2023) A growing
understanding of the role of muscarinic
receptors in the molecular pathology
and treatment of schizophrenia.
Front. Cell. Neurosci. 17:1124333.
doi: 10.3389/fncel.2023.1124333
COPYRIGHT© 2023 Dean, Bakker, Ueda, Tobin, Brown and
Kanaan. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in otherforums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in thisjournal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does notcomply with these terms.

Synaptic Biology and Cognition Laboratory, The Florey Institute of Neuroscience and Mental Health,
Parkville, VIC, Australia, 2 Sosei Heptares, Cambridge, United Kingdom, 3 Department of Systems
Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 4 Laboratory for
Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan, 5 Advanced ResearchCentre (ARC), School of Molecular Bioscience, University of Glasgow, Glasgow, United Kingdom,
6
Department of Psychiatry, Austin Health, The University of Melbourne, Heidelberg, VIC, Australia

Pre-clinical models, postmortem and neuroimaging studies all support a role formuscarinic receptors in the molecular pathology of schizophrenia. From thesedata it was proposed that activation of the muscarinic M1 and/or M4 receptor
would reduce the severity of the symptoms of schizophrenia. This hypothesis is
now supported by results from two clinical trials which indicate that activating
central muscarinic M1 and M4 receptors can reduce the severity of positive,
negative and cognitive symptoms of the disorder. This review will provide an
update on a growing body of evidence that argues the muscarinic M1 andM4 receptors have critical roles in CNS functions that are dysregulated by thepathophysiology of schizophrenia. This realization has been made possible, inpart, by the growing ability to visualize and quantify muscarinic M1 and M4
receptors in the human CNS using molecular neuroimaging. We will discuss how
these advances have provided evidence to support the notion that there is a
sub-group of patients within the syndrome of schizophrenia that have a uniquemolecular pathology driven by a marked loss of muscarinic M1 receptors. This
review is timely, as drugs targeting muscarinic receptors approach clinical use forthe treatment of schizophrenia and here we outline the background biology that
supported development of such drugs to treat the disorder.
KEYWORDS

muscarinic M1 receptor, muscarinic M4 receptor,
neuroimaging, postmortem CNS, xanomeline, KarXT

schizophrenia,

sub-group,

1. Introduction
The reports of a successful phase III trial (Lahucik, 2022) and a large phase II
trial (Brannan et al., 2021), suggest a coformulation of xanomeline and trospium canreduce the symptoms of schizophrenia without causing unacceptable side-effects. The useof xanomeline, a partial agonist with preference for muscarinic M1 and M4 receptors

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Dean et al.

10.3389/fncel.2023.1124333(CHRM1/CHRM4)1 (Bymaster et al., 1997), to treat schizophrenia
was underpinned by a hypothesis reflecting many lines ofevidence arguing that activating CHRM1 and CHRM4 would
be a mechanism to reduce the severity of the symptoms ofthe disorder (Bymaster et al., 1999; Dean et al., 2003). These
exciting new developments around targeting CHRM1 and 4 to treat
schizophrenia mean it is timely to review the role for CHRMs in
the molecular pathology and treatment of the disorder. The notionof targeting CHRMs in the clinical management of schizophrenia is
not new as pan-CHRM antagonists were used extensively to control
the extrapyramidal side effects of first-generation antipsychotic
drugs (Montastruc et al., 2018). With regards to the symptoms ofschizophrenia, it is notable that treatment with high affinity CHRM
antagonists to control extrapyramidal side effects worsened thecognitive deficits in people with the disorder (Tracy et al., 2001).
These data show that improving the side effects of antipsychotic
drugs are not necessarily linked to improving clinical outcomes forpeople with schizophrenia.
Recent progress in developing drugs that target CHRMs
to treat schizophrenia has occurred because of an increasing
understanding of the molecular pathology of the disorder thatsuggested therapeutically beneficial drugs needed to activate, notinhibit, CHRMs (Bymaster et al., 2002; Dean et al., 2003). Moreover,such drugs needed to target specific, not all, CHRMs so as togive different biochemical and behavioral outcomes that would
be beneficial when used to treat schizophrenia; the developmentof such drugs has been comprehensively reviewed elsewhere (e.g.
(Felder et al., 2018; van der Westhuizen et al., 2020; Foster et al.,2021; Johnson et al., 2022). Hence, this review will focus on the data
supporting a role for the CHRM1 and CHRM4 in the molecularpathology of schizophrenia and the physiological processes thatare affected by the changes in CHRM1 and CHRM4 signaling thatwould be postulated to be occurring in people with the disorder.

compounds that could activate or inhibit individual receptors innative tissue.
The distribution of CHRMs also plays a critical role in theoutcome of each receptor being activated by acetylcholine. CHRM1
has a widespread distribution throughout the CNS but its levels
are highest in cortical regions and the hippocampus, at times
constituting more than 40% of total CHRMs (Flynn et al., 1995).
Importantly, the CHRM1 is mainly located post-synaptically on
excitatory neurons (Mrzljak et al., 1993) and, although present
across the whole cortex, the CHRM1 is most prominent in corticallayers III and V/VI (Harrison et al., 1991; Scarr et al., 2018a). The
CHRM1 is predominantly located on pyramidal neurons in thecortex (Zhang et al., 2002; Scarr et al., 2018a) and hippocampus(Scarr et al., 2016a). The CHRM2 is highly expressed in the
nucleus basalis and occipital cortex and is present at lower levelsin the hippocampus, caudate putamen and other cortical regions(Flynn et al., 1995). Significantly, in the cortex the CHRM2 islocated pre- and post-synaptically (Mrzljak et al., 1993), the presynaptic CHRM2 acting as auto-receptors aiding in controllingthe synthesis and release of acetylcholine (Zhang et al., 2002).
By contrast, the post-synaptic CHRM2 is present on excitatory
synapses (Mrzljak et al., 1998) with close to a third of cortical
GABAergic neurons expressing CHRM2 (Disney and Aoki, 2008).
The distribution of CHRM3 is similar to CHRM1 but the level ofthat receptor is much lower than CHRM1 and, at least in the rat,
the CHRM3 is reported to be present on glia as well as corticalpyramidal neurons (Levey et al., 1994). Levels of CHRM4 arehighest in the striatum but make up approximately 20% of thetotal CHRMs in the cortex and hippocampus (Flynn et al., 1995).
In the striatum the CHRM4 is often associated with dopaminergic
receptors (Weiner et al., 1990) and it is suggested the CHRM4
is critical in the mechanism by which acetylcholine modulates
dopamine transmission in the striatum and / or nucleus accumbens(Threlfell and Cragg, 2011). The CHRM5 has only been detected
at very low levels in hippocampus, substantia nigra, and ventral
tegmental area (∼ 5% of total CHRMs) (Vilaro et al., 1990). One
outcome of this differential distribution of CHRMs is that different
receptors can differentially respond to acetylcholine to modulate
the many effects of that neurotransmitter across the CNS (Perryet al., 1999).
A greater understanding of the role of individual CHRMs in
the physiology of the whole organism was achieved following thedevelopment of mice that did not express the gene for one, or
multiple, CHRMs (Wess, 2004). Here, because of the focus on
schizophrenia, we will review the data pertinent to CHRM1 andCHRM4 as these are now implicated in the molecular pathology
and treatment of the disorder.

2. CHRM1 and CHRM4 receptor
functions: Studies using receptor
knockout mice
DNA sequencing technologies established that, in mammals,
there were 5 genes encoding different CHRMs (Liao et al., 1989).
These receptors mediate a variety of physiological outcomes
because they regulate different signaling pathways and are
differentially distributed across tissues and cell types (Felder, 1995).
The family of CHRMs consists of 5 receptors (designated CHRM1,
CHRM2, CHRM3, CHRM4 and CHRM5) with CHRM 1, 3 and5 modulating the activity of signaling pathways that include
phospholipases A2, C, and D, as well as tyrosine kinase and anovel class of voltage-insensitive calcium channels. By contrast,
CHRM 2 and 4 were shown to modulate phospholipase A2 andadenylate cyclase activity. These findings were initially based on theavailability of cloned receptors expressed in cell culture; by contrast,
studies in intact animals were limited because of the absence of

2.1. CHRM1Using mice that did not express functional CHRM1, it was
shown that receptor had a role in activating the mitogen-activatedprotein kinase (MAPK) pathway, a pathway that has critical roles
in synaptic plasticity and maintaining cognitive ability (Wesset al., 2007). Significantly, whilst several studies confirmed thepresence of deficits in neural plasticity in the forebrain of theCHRM1−/− , initial behavioral studies did not show clear deficits
in their cognitive abilities (Miyakawa et al., 2001). This was because

1 For ease of interpretation, in this review the abbreviation for muscarinic
receptors will be consistently capitalized rather than alternating to upperand lower-case abbreviations normal when citing studies in species
other than humans and will be used instead of the recognized protein
abbreviations ending in mAChR.
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the behaviors studied, such as matching-to-sample tasks, involved
hippocampal learning that did not require CHRM1 signaling.
It was subsequently shown that whilst CHRM1−/− mice hadvery small changes in hippocampal long-term potentiation, they
had significant deficits in tasks requiring non-matching-to-sample
working memory and consolidation (Anagnostaras et al., 2003).
These findings allowed the conclusion that the CHRM1 was criticalin maintaining cognitive-related functions that required corticalengagement.
Ongoing studies of the cortical CHRM1 have shown that
CHRM1 agonist-stimulated activation of mitogen-activatedprotein kinases (MAPK) is essentially abolished, and agoniststimulated phosphatidyl inositol (PI) hydrolysis is reduced by>60%, in primary cortical cells from newborn CHRM1−/− mice
(Hamilton and Nathanson, 2001). Going beyond well-defined
CHRM signaling pathways, HEK293 cells were used to show
CHRM1 signaling effects gene expression (von der Kammer et al.,2001). This effect of CHRM1 signaling has been further studied in
the CHRM1−/− mouse where there are extensive changes in gene
expression that would affect many aspects of cortical functioning(Supplementary Figure 1; Dean and Scarr, 2021). The challengethat remains is to identify which changes in these functions are
responsible for the cortical-mediated behavioral changes in the
CHRM1−/− mouse.
At the sub-cortical level, the CHRM1−/− mouse has a twofold increase in levels of extracellular dopamine in the striatum(Gerber et al., 2001) which could be responsible for its pronounced
increase in locomotor activity (Miyakawa et al., 2001). Further
changes in behaviors and physiology in the CHRM1−/− mouse
have highlighted the important role of that receptor outside
the CNS, notably counteracting bronchoconstriction, possibly
by releasing a relaxing agent stored in respiratory epithelia or
pulmonary nerves (Struckmann et al., 2003). This is unlikely to be
the only peripherally mediated CHRM1 effect given the receptor
has been shown to be expressed by cells in the cardiovascularsystem (Saternos et al., 2018), the lung (Haddad et al., 1996),
gastric mucosa, skin, melanocytes and immunocytes (Schledwitz
et al., 2021). Hence, as it is increasingly recognized the molecularpathology of diseases of the human CNS extend into the periphery(Scarr et al., 2015; Lai et al., 2016b), it will be important toconsider if CHRM1-mediated pathophysiology in disorders such as
schizophrenia also extends beyond the CNS.

locomotor activity (a mouse behavior associated with increased
dopaminergic activity) in the CHRM4−/− mouse (Gomeza et al.,1999). These data all support the notion that the absence of theCHRM4 leads to increased dopaminergic activity in the CNS
which is also argued to be involved in the genesis of psychotic
symptoms in people with schizophrenia (Carlsson, 1977). Thisis therefore a biological basis for the suggestion that specifically
activating the CHRM4 would be a mechanism to reduce thepsychotic symptoms of schizophrenia (Bymaster et al., 2002). Thishypothesis has gained support from a recent trial that has reported
that the CHRM4 positive allosteric modulator (PAM), emraclidine,
reduces the severity of both acute psychotic and acute negative
symptoms in people with schizophrenia (Krystal et al., 2022).
Importantly, all receptor-specific CHRM PAMs increase the activityof a single CHRM by binding to an allosteric binding site toincrease the responsiveness of the receptor to acetylcholine byincreasing the probability that the receptor will be activated by theneurotransmitter (Yohn and Conn, 2018).
It has also been reported that the enhancement of acetylcholine
efflux caused by the pan-CHRM antagonist, scopolamine, wasmarkedly reduced in the CHRM4−/− mouse mid-brain (Tzavara
et al., 2004). These data show that the CHRM4 has criticalroles in controlling both dopaminergic and cholinergic activity
in sub-cortical regions. This could be why there was an absenceof scopolamine-mediated suppression of haloperidol-induced
catalepsy in the CHRM4−/− mouse (Karasawa et al., 2003; FinkJensen et al., 2011). These data suggest that, in addition to having
the potential for direct effects on levels of psychotic symptoms(Krystal et al., 2022), activation of the CHRM4 may also act tolessen some of the unwanted effects of the dopamine D2 receptor
antagonist drugs that are the mainstay of treating psychosis.
Outside the CNS, there is now preliminary evidence to show
expression of CHRM4 in peripheral tissue, including the gastricmucosa, small intestine, skin, melanocytes and immunocytes(Schledwitz et al., 2021). In this regard, studies using CHRM4−/−
have shown CHRM4 has an important role in mediating skinkeratinocyte adhesion (Nguyen et al., 2004) in a way that facilitateswound repair through keratinocyte migration and wound reepithelialization (Chernyavsky et al., 2004).
Until the availability of receptor knockout mice, due to theabsence of CHRM receptor-specific drugs, it was impossible toallocate changes in specific behaviors or biochemical pathways toan individual CHRM. Hence the data from the CHRM1−/− andCHRM4−/− gave the first real insight into the diverse impact
CHRM1 and CHRM4 signaling had on CNS function at themolecular and behavioral level (Wess et al., 2006). However, thesynthesis of drugs that can specifically target individual CHRMs(Conn et al., 2009) and the development of new technologies, suchas optogenetics (Deisseroth et al., 2006), have allowed alternative
approaches to understanding the functions of CHRMs in the CNS
and in the periphery.

2.2. CHRM4
Findings using the CHRM4−/− mouse have emphasized thestrong interactions of the CHRM4 and the glutamatergic systems
as those mice had increased sensitivity to the disruption of prepulse inhibition (PPI) following N-methyl-D-aspartate (NMDAR)
receptor blockade by phencyclidine (Felder et al., 2001). These
data are significant because abnormal control of PPI has beensuggested to be a useful biomarker for schizophrenia (Mena et al.,2016). In addition, CHRM4−/− mice have markedly increased
basal dopamine levels in the nucleus accumbens, with bothd-amphetamine and phencyclidine causing a greater increase in
dopamine efflux compared to wild type mice (Tzavara et al.,2004). Finally, there was a small, but significant, increase in basal

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2.3. Muscarinic M1 and M4 receptor
signaling
The power of newer approaches to studying the cholinergic
system in the CNS is demonstrated by a study that used optogenetic

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striatum, thalamus and intergeniculate leaflet causes changes to the
biological rhythm of locomotor activity in mice (Riljak et al., 2020).

stimulation to cause a transient increase in acetylcholine
release in the striatum, which caused a long-lasting increasein excitability of medium spiny neurons that was associatedwith a hyperpolarizing shift of action potential thresholds anda decreased hyperpolarization amplitude (Lv et al., 2017). The
outcome of this was an increase in probability of excitatory
postsynaptic potential-action potential coupling. It was thenshown that CHRM1 selective antagonist prevented the ability ofa CHRM1 selective positive allosteric modulator to potentiate
an acetylcholine-mediated persistent increase in medium spiny
neuron intrinsic excitability. This combined use of optogenetics
and receptor-selective and specific drugs has therefore revealed theimportance of the CHRM1 in mediating the function of medium
spiny neurons in the striatum.
Critical to understanding the role of CHRMs in CNS
functioning is an understanding of how these receptors modulate
other neurotransmitters systems. It was therefore significant thatstudies localizing RNA to cells in the primate CNS show that the
CHRM1 is located on extrinsic glutamatergic and monoaminergic
afferents as well as intrinsic GABAergic afferents (Alcantara et al.,2001). These data have now been expanded to suggest potentialfunctional outcomes from interactions between the CHRM1 andthe glutamatergic system in the CNS. Hence, it has been shown thathippocampal CHRM1 facilitates cognitive function by interplay
with the AMPA receptor GluA1 subunit (Zhao et al., 2018). In
addition, it has been shown that CHRM1 modulation of the
AMPA GluA1 receptor sub-unit involves signaling through protein
kinase C and Ras (Chen et al., 2020), possibly by differential
phosphorylation of the AMPA GluA1 receptor sub-unit (Zhaoet al., 2019b). This pathway may have some clinical significance, as
it is suggested that through this process CHRM1 activation could
rescue β-amyloid mediated cognitive impairment (Zhao et al.,2019a), is an important pathophysiological process associated with
Alzheimer’s disease (Hardy and Higgins, 1992).
As is the case for interactions between the CHRM1 andglutamatergic systems, recent receptor-specific studies continue
to argue for a role of the CHRM1 in modulating cognition. For
example, modulating CHRM1 signaling, using the receptor specificdrug VU0453595, has been shown to enhance the performanceof mice in cue-dependent water-based T-maze, a dorsolateral
striatum-dependent learning task (Lv et al., 2017). However, thismay not be a simple linear relationship, as it has been reported
that overstimulation of the CHRM1 in the primate prefrontal
cortex can disrupt working memory (Vijayraghavan et al., 2018).
In the primate cortex, and in rodent cortex, CHRM1 is known
to modulate working memory via multiple pathways that include
the N-methyl-D-aspartate receptor (Galvin et al., 2021) and KCNQ
potassium channels (Galvin et al., 2020). It would therefore be
worthwhile to determine if any or all of these mechanisms become
non-responsive after overstimulation of the CHRM1.
Whilst studies focused on the CHRM4 may be less numerous,
it has been reported that activating that receptor modulates bothacquisition and consolidation of memory functions (Gould et al.,2018). In addition, striatal and nigral CHRM4 signaling leads tomultilevel inhibition of striato-nigral pathways which can result in
an attenuation of dyskinesia (Brugnoli et al., 2020), which links theCHRM4 to dopaminergic activity in the CNS (Porras et al., 2014).
In addition, it has recently been shown that the CHRM4 in the

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2.4. Muscarinic receptors and REM andnon-REM sleep
It has long been known that multiple neurotransmitter systems
are involved in regulating REM sleep (Fraigne et al., 2015;
Falup-Pecurariu et al., 2021) with cholinergic neurotransmission
being important in controlling REM sleep (Yamada and Ueda,
2020). Due to the focus of our review we will provide
commentary on a growing understanding of the role of CHRM1
in regulating REM sleep because a broader review of the roleof the cholinergic system in REM sleep is available elsewhere(Yamada and Ueda, 2020).
Like many aspects of understanding the mechanisms by whichacetylcholine can influence physiological outcomes, understanding
how acetylcholine influences sleep has been hampered by theabsence of appropriate experimental tools. However, with thedevelopment of technologies such as triple-CRISPR and ES-mice(Sunagawa et al., 2016; Ukai et al., 2017) it is now possible tounderstand the mechanisms by which acetylcholine can control
sleep. Hence, it has now been shown that the CHRM1−/− /CHRM3−/− double knockout mouse has an enrichment of EEG
theta oscillation during sleep, leaving the theta oscillation largely
unaffected during wakefulness (Niwa et al., 2018). This study
advanced earlier findings that showed pan-muscarinic antagonists
such as atropine or scopolamine reduce the theta oscillationin anesthetized animals (Kim and Jeong, 1999; Buzsaki, 2002)
to suggest the CHRM1 and CHRM3 are critically involvedin controlling REM. Moreover, extensive use of newer genemodification techniques has shown that nicotinic acetylcholine
receptors failed to affect sleep phenotypes (Niwa et al., 2018).
In addition, it has been shown that CHRM1−/− mice haveshorter REM sleep duration, but only slightly shorter NREM
sleep duration, whilst CHRM3−/− mice have shorter NREM sleep
duration, but their REM sleep duration was comparable to thatof WT mice (Niwa et al., 2018). These findings suggest further
studies are needed to better understand the molecular and cellularmechanisms involved in these receptor specific phenotypes.
Without direct experimental evidence, it is of interest that
CHRM1 and CHRM3 make up roughly 60 and 10% of the total
muscarinic acetylcholine receptors in the hippocampus (Dasari
and Gulledge, 2011), respectively. This could be important because
slow-wave and delta oscillations originate from the neocortex andthe thalamus, respectively, whereas theta oscillation is thought tooriginate from the hippocampus (Sirota et al., 2008). Hence thedifferential distribution of CHRM1 and CHRM3 across the regions
and different cells in the hippocampus may be critical to theireffect on sleep. However, before these data are available it will
be of great interest to see if drugs such as KarXT, when used totreat schizophrenia, may improve the sleep disturbances associatedwith the disorder. Such an outcome would seem possible given
the recent report that the CHRM1 positive allosteric modulator,
VU0453595, has been shown to modulate arousal and sleep / wakearchitecture in young and aged rodents, and nonhuman primates(Gould et al., 2020).

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2.5. Muscarinic receptors andschizophrenia: Postmortem CNS studies(Mcomish et al., 2017) or increased (Lange et al., 1993) in
Parkinson’s disease.
Understanding the mechanisms causing lower levels of cortical[3 H]pirenzepine binding has been advanced by studies showing
lower levels of CHRM1 gene expression (Dean et al., 2002;
Mancama et al., 2003) and protein in the DFPLC of people withschizophrenia. By contrast, it has been reported that levels of
CHRM2, CHRM3 (Scarr et al., 2006) and CHRM4 (Dean et al.,2002) were not altered in the DFPLC from those with the disorder.
In addition, changes in CHRM1 (Salah-Uddin et al., 2009), but not
CHRM2/4, signaling has been reported in the DFPLC in people
with schizophrenia.
Hence, current data would argue that changes in CHRM1 inthe cortex, but not necessarily other CNS regions, are a significant
component of the molecular pathology of schizophrenia.

The first evidence suggesting there were changes in levelsof CHRMs in people with schizophrenia was generated using
postmortem CNS and radioligand binding. Using such an
approach, it was reported that levels of [3 H]pirenzepine binding
were lower in the striatum (Dean and Crook, 1996), dorsolateralprefrontal cortex (DLPFC) (Crook et al., 2001), the anterior(Zavitsanou et al., 2004) and posterior cingulate cortex (Newell
et al., 2007), superior temporal gyrus (Deng and Huang, 2005),
premotor cortex (Dean et al., 2008b) and hippocampus (Crooket al., 2000) compared to non-psychiatric controls. Notably,compared to controls, [3 H]pirenzepine binding was not alteredin the thalamus of people with schizophrenia (Dean et al., 2004).
In interpreting these results, it is important to note the various
components that contribute to the selectivity of any radioligand
binding assay. In the case of our radioligand binding assay it was
important that the use of cloned CHRMs showed that unlabeledpirenzepine bound with high affinity to the CHRM1 and with aslightly higher affinity than it did to other CHRMs as the drug
had 6, 7, 13 and 17 fold lower affinity for CHRM4, CHRM5,
CHRM3 and CHRM2, respectively (Buckley et al., 1989). These
data indicate the use of pirenzepine gives any assay a slightselectivity for the CHRM1. However, the overall selectivity of aradioligand binding assay depends on the selective binding of
the radioligand and the displacing agent used to define nonspecific binding (Bylund and Toews, 1993). In developing ourradioligand binding assay we used unlabeled drugs that wereselective for CHRM1 such as quinuclidinyl xanthene-9 -carboxylate
hemioxilate (Crook, 1998) or 3-quinuclidinyl benzilate (Stanton
et al., 1993) to increase selectivity for that receptor. Finally,
we showed that the on-rate of [3 H]pirenzepine binding for the
CHRM1, as demonstrated by the binding of that radioligand tohuman cloned CHRM1 and CHRM4 (Scarr and Dean, 2008),
was considerably faster than for the CHRM4 which means thatmeasuring [3 H]pirenzepine binding after 30 min incubations
results in the radioligand binding being highly selective for the
CHRM1. The selectivity of binding under these conditions havebeen confirmed using tissue from mice lacking CHRM1 or CHRM4(Scarr and Dean, 2008; Gibbons et al., 2013), components of snake
venom that show strong selectivity for the CHRM4 and CHRM4
specific allosteric modulators (Valuskova et al., 2018). Hence, thestudies showing lower levels of [3 H]pirenzepine binding in people
with schizophrenia where the incubation time was short (Deanet al., 1996, 2008b; Crook et al., 2000, 2001) argue the decreased
radioligand binding would be due to lower levels of CHRM1. Bycontrast, decreased [3 H]pirenzepine binding in the studies using alonger incubation (Zavitsanou et al., 2004; Deng and Huang, 2005;
Newell et al., 2007) could be due to lower levels of either receptor
in the CNS of people with schizophrenia.
It is of significance that lower levels of cortical [3 H]pirenzepine
binding in schizophrenia shows some diagnostic specificity. Thisis because, compared to controls, cortical [3 H]pirenzepine binding
was not different in the cortex of people with major depressive
disorders (Zavitsanou et al., 2004; Gibbons et al., 2009), bipolar
disorders, and Alzheimer’s disease (Scarr et al., 2017). In addition,
cortical [3 H]pirenzepine binding has been reported as unaltered

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2.6. The CHRM1 and the syndrome ofschizophrenia
Schizophrenia is recognized as being a syndrome of disorders
which present with the same defining constellation of symptoms(Jablensky, 2010) and it is now recognized that dissecting the
syndrome into more homogeneous sub-groups is critical toadvancing understanding of the pathophysiology of the syndrome
and its components (Jablensky, 2006; Tamminga et al., 2017).
Hence, it is significant that approximately 25% of people withschizophrenia could be separated into a sub-group, termed the
CHRM deficit sub-group (MRDS), because they have a marked lossof cortical CHRM1 (Scarr et al., 2009).
The identification of MRDS has enabled studies to compare
its molecular pathology to schizophrenia where there is no
determinable loss of cortical CHRM1 (non-MRDS). This approach
has shown that those with MRDS have no increased association
with any sequence variation within the CHRM1 gene or its
promotor (Scarr et al., 2009), exposure to antipsychotic drugs(Dean et al., 2015, 2016a, 2008b; Scarr et al., 2018a,c), theanticholinergic load in the cortex (Dean et al., 2020) or smoking
status in MRDS compared to non-MRDS. By contrast, a numberof changes in biochemical homeostasis comparing MRDS andnon-MRDS have been identified (Table 1). For example, there
are differential changes in gene expression (Scarr et al., 2018c)
and levels of micro-RNAs controlling CHRM1 gene expression
and translation (Scarr et al., 2013a) in MRDS compared to nonMRDS. By contrast, changes in levels of transcription factors
regulating CHRM1 expression did not differ between MRDS andnon-MRDS (Seo et al., 2014; Scarr et al., 2018c). This proposedmechanism is supported by data showing that, compared tocontrols and people with non-MRDS, those with MRDS do nothave changes in innervating cholinergic neurons as indicated bylevels of choline acetyltransferase (Dean et al., 2020) but have lower
levels of CHRM1 in many cortical areas (Gibbons et al., 2013; Seoet al., 2014), lower responsiveness to CHRM1 allosteric modulation(Dean et al., 2016a; Hopper et al., 2019), and higher levels of the α7nicotinic receptor (Dean et al., 2020; Table 1). The higher levels of
α7 nicotinic receptor in people with MRDS argues that the subgroup may have a breakdown in cholinergic homeostasis that is
not limited to the CHRM1 and CHRM4, and that MRDS may

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TABLE 1 A summary of differential findings relating to the molecularpathology of a sub-group of people with schizophrenia and a marked
loss of cortical CHRM1 (MRDS) and other people with schizophrenia who
do not have a marked loss of cortical CHRM1 (non-MRDS)
compared to controls.

be particularly responsive to being treated with drugs that canselectively target the α7 nicotinic receptor [as argued by Jones et al.
(2012)] and the CHRM1.
The notion of sub-group specific changes in CHRM1-related
biochemistry has recently been suggested to account for changes in
the coupling of CHRM1 to Gα q/11 in the dorsolateral prefrontal
cortex of people with schizophrenia (Odagaki et al., 2020). Thissuggestion adds to an earlier report showing that abnormalities in
CHRM1-coupling to Gα q/11 was altered in people with MRDS but
not in non-MRDS (Salah-Uddin et al., 2009). By contrast, using
tissue from the same cases, CHRM1-coupling to Gα q/11 was notaltered in MRDS or non-MRDS when the receptor was activatedusing the selective CHRM1 allosteric agonist AC-42 (Salah-Uddin
et al., 2009). Notably, it is recognized that the CHRM1 can undergo
conformational changes depending on the receptor occupancy state
at different sites on the receptor (Maeda et al., 2019). It is therefore
intriguing to postulate whether the orthosteric binding signaling inschizophrenia and MRDS could be due to some intrinsic modulatorof receptor binding as has been suggested for the GABAA receptor(van Kammen et al., 1993) and the serotonin 2A receptor (Deanet al., 2008a).
When considering the breakdown in cholinergic homeostasis
in schizophrenia it is significant that people with MRDS and nonMRDS have lower levels of CHRM1-expressing pyramidal cells inlaminae III and V of the DLPFC (Scarr et al., 2018a). Notably,
this loss of CHRM1-positive pyramidal cells occurred without any
changes in total neuron and glia cell number (Scarr et al., 2018a)
or in changes in levels of pre-synaptic boutons as measured using
synaptosome nerve-associated protein 25 (Dean et al., 2020). These
data strongly argue there must be aberrant CHRM1 signaling inboth MRDS and non-MRDS because fewer pyramidal cells will
respond to stimulation by acetylcholine. There are also otherbiochemical changes common to both MRDS and non-MRDS,
which include higher levels of cortical RNA encoding the zinctransporter SLC39A12 (Scarr et al., 2016b), selenium binding
protein (Udawela et al., 2015) and lower levels of phospholipase C
beta 1 RNA (Udawela et al., 2017). In addition, people with nonMRDS have been shown to have changes in levels of pyruvate kinase
in the striatum (Table 1), an enzyme which is critically linked tothe activity of the Embden-Meyerhof pathway (Dean et al., 2016b).
These data are of interest because neuroimaging studies identified a
sub-group of patients with schizophrenia that had abnormalities instriatal glucose utilization (Buchsbaum et al., 1992); it is intriguing
to postulate whether this was due to abnormal glucose utilization
due to the altered levels of pyruvate kinase observe in non-MRDS.
Whilst there is a growing understanding of disturbances incortical homeostasis in MRDS and non-MRDS, recent data shows
there are lower levels of CHRM1 in the hippocampus and striatumin those in the MRDS sub-group (Dean et al., 2016a; Hopper et al.,2019). Hence further studies comparing changes in hippocampal
and sub-cortical molecular pathology will need to be conducted to
fully understand the pathophysiology of MRDS.
Whilst it has long been argued that CHRM1 plays an importantrole in the pathology of schizophrenia (Raedler et al., 2007), recent
findings suggest the receptor is important in regulating diverse
functions that are affected in those with the disorder. For example,
the CHRM1 and CHRM3 have been shown to be important inodor discrimination and learning (Chan et al., 2017), a deficit thathas been demonstrated in people with schizophrenia (Brewer et al.,2001). It has also been shown that 19 -tetrahydrocannabinol, given
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MRDS
Dorsolateralprefrontal cortex

Non-MRDS

Markedly lower level of CHRM1
in multiple cortical regions.
Unique changes in gene
expression in the dorsolateralprefrontal cortex.
Higher levels of α7 nicotinic
receptors in the dorsolateralprefrontal cortex.
Changed CHRM1 promotor
methylation.
Higher levels of the microRNA
107.
Lower numbers of CHRM1
positive neurons in laminae III
and IV and lower levels of
CHRM1 in the dorsolateralprefrontal cortex.

Lower number of CHRM1 positive
neurons in laminae III and IV withunchanged levels of CHRM1 inthe dorsolateral prefrontal cortex.

Unchanged level of innervatingcholinergic neurons in thedorsolateral prefrontal cortex.

Unchanged level of innervatingcholinergic neurons in thedorsolateral prefrontal cortex.

Higher level of RNA encoding thezinc transporter SLC39A12.

Higher level of RNA encoding thezinc transporter SLC39A12.

Higher level of RNA encoding
selenium binding protein.

Higher level of RNA encoding
selenium binding protein.

Lower level of RNA encoding
phospholipase C beta 1.

Lower level of RNA encoding
phospholipase C beta 1.

Hippocampus

Markedly lower level of CHRM1.

StriatumMarkedly lower level of CHRM1.
Changes in pyruvate kinase,pyruvate and lactate consistent
with an inability to process
glucose through the
Embden-Meyerhof-Parnas
pathway.

to rats during adolescence, decreases the expression and functionof cortical CHRM1 (Garzon et al., 2021). This is significant given
the relatively high use of cannabis by people with schizophrenia(Winklbaur et al., 2006). People with schizophrenia are known tohave an increased risk of sleep disturbance (Kaskie et al., 2017).
Importantly, sleep is a cyclic process that is composed of rapid
eye movement (REM) and non-rapid eye movement (NREM) sleep(Pocivavsek and Rowland, 2017). In schizophrenia, a number ofstudies have reported reductions in the total time sleeping and agreater latency to fall asleep, decreases in REM latency and duration
as well as reductions in total NREM (Pocivavsek and Rowland,2017). Pertinent to this review, it is known that acetylcholine is
crucial in controlling REM sleep (Hobson et al., 1975; Sakai et al.,2001).

2.7. CHRM4 and the molecular pathology
of schizophrenia
Current data suggests that levels of CHRM4 are not altered in
the prefrontal cortex or parietal cortex of people with schizophrenia(Dean et al., 2002). However, the neocortex contains diverse and06

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functioning (Holland et al., 2021). Hence it is likely that COMT
could affect cognition by affecting levels of both dopamineand noradrenaline. Significantly the association between COMT
genotype and levels of CHRM1 was linked to two genotypes(rs4680 and rs4818) (Dean and Scarr, 2016). This is significant
because rs4818 is linked to cognitive ability in humans (Roussos
et al., 2008) but this association does not primarily involve themodulation of the catecholaminergic system, rather it is due toand alteration in the structure of COMT RNA which results inaltered levels of translation of the RNA to protein (Nackley et al.,2006). This mechanism, as well as the modulation in catecholamine
activity, could be modulating the link between COMT genotype
and cognition in humans. This mechanism is of interest because
it has been reported that that genotype at rs4680 and rs4818 areassociated with differing levels of soluble COMT (S-COMT), but
not membrane-bound COMT (MB-COMT), in the human cortex(Parkin et al., 2018). This finding is relevant because MBCOMT,
which is localized on the cell membrane and present on cell bodies,axons and dendrites of neurons, has a role in the breakdown of
the catecholamines (Chen et al., 2011) but S-COMT is localized
in the cytoplasm where it has access to many more catechol-based
substrates that include the catechol estrogens (Schutze et al., 1994).
This means that S-COMT can impact on cellular functioning byaltering levels of catechol estrogens which will affect the occupancy
and nuclear localization of estrogen receptors (Schutze et al., 1994;
Levin, 2001) and thereby alter levels of the expression of genesthat have estrogen response elements (Klinge, 2001). Hence, thelink between levels of CHRM1 expression and COMT genotypes
likely involves the actions of S-COMT via its ability to indirectly
modulate gene expression which could be either an alternative orcooperative pathway to the ability of M-COMT to modulate thefunctioning of dopamine and noradrenaline (Parkin et al., 2018).
There may also be a role for the CHRM4 gene as a susceptibility
gene for schizophrenia, as it has been reported that the C/C allele
at the 1341 single nucleotide polymorphism carries an increased
risk for schizophrenia that is resistant to treatment with current
drugs (Scarr et al., 2013b). More recently, it has been suggested
that the T/T allele at the same single nucleotide polymorphism
was associated with an increased risk of developing schizophrenia(Pozhidaev et al., 2020). It is not readily apparent why such different
outcomes could occur, but there are significant methodologicaldifferences between the studies that includes using cohorts withdifferent genetic backgrounds. This could be important as a study
comparing cortical [3 H]pirenzepine-binding in Japanese people
with schizophrenia to controls from Australia failed to show any
differences between the diagnostic cohorts (Matsumoto et al.,2005).
Despite these potentially interesting findings, it must be
acknowledged that no strong association between CHRMs genesequence variation and schizophrenia has been established in thegrowing large GWAS database (Dennison et al., 2020).

separate regions in which biochemical homeostasis is differentiallyaffected by the molecular pathology of the disorder (Scarr et al.,2018b). It therefore remains possible that changes in cortical
CHRM4 levels have yet to be identified within some corticalregions. It is also noteworthy that there are decreased levels of[3 H]pirenzepine binding across many regions of the hippocampus(Crook et al., 2000; Scarr et al., 2007). One of these studies also
reported lower levels CHRM4, but not CHRM1, mRNA acrossthe hippocampus from people with schizophrenia compared tocontrols (Scarr et al., 2007). These data would argue that at least
some of the decrease in [3 H]pirenzepine binding in schizophrenia
was due to low levels of CHRM4. In the striatum, it has beenreported that levels of [3 H]pirenzepine binding (Dean et al., 1996)
but not CHRM1 or CHRM2 mRNA (Dean et al., 2000), werelower in people with schizophrenia compared to controls. These
data would favor the argument that lower levels of CHRM4 were
contributing to the lower levels of [3 H]pirenzepine binding in the
striatum of people with schizophrenia. However, it is clear morestudies on CHRMs in the hippocampus and striatum are warranted
to get a clear understanding about the changes in those receptors inschizophrenia.

2.8. Genetics, muscarinic receptors, andcognition in schizophrenia
It is now widely accepted that schizophrenia occurs
in individuals with a genetic susceptibility after adverse
environmental exposure (Smigielski et al., 2020). With regards toCHRM1 levels in people with schizophrenia, current data suggeststhat the lower levels of CHRM1 are not associated with a genevariant (Scarr et al., 2009) but could be due to changes in gene
methylation (Scarr et al., 2013a) which is affected by environmental
factors (Smigielski et al., 2020). However, based on findings using
DNA from blood, there may be an association between genotype at
a 267C/C single nucleotide polymorphism in the CHRM1 gene andcognitive functioning in schizophrenia (Scarr et al., 2012).
As well as a direct link between the CHRM1 gene sequenceand cognition, it has also been reported that low levels of cortical
CHRM1 are associated with single nucleotide polymorphisms in
the catechol-O-methyltransferase (COMT) gene that are associatedwith poorer cognitive ability (Dean and Scarr, 2016). These data
would suggest that people with schizophrenia who have lower
levels of CHRM1 could, as has previously been suggested (Eganet al., 2001), have cognitive deficits that are at least in partdue to a breakdown in homeostasis in pathways regulated by
COMT. Initially it was postulated that the linked between COMT
genotype at rs4680 (valine to methionine substitution: Val 108/158
Met) and cognition was due to the methionine substitute COMT
being less able to metabolize dopamine leading to an increasein the storage and release of dopamine from the pre-synaptic
neuron (Weinshilboum et al., 1999). This increased release of
dopamine was important because of evidence to suggest that
increased dopaminergic activity in the prefrontal cortex was linked
to levels of working memory (Goldman-Rakic, 1997). However,it has been shown that COMT can affect the metabolism ofboth dopamine and noradrenaline (Eisenhofer and Finberg, 1994)
and noradrenaline has also been shown to modulate cognitive

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2.9. Muscarinic receptor molecularimaging studies in schizophrenia
Studies investigating levels of CHRMs and their relationshipto CNS function in people with schizophrenia have been limited

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the two studies, the study showing lower 123 I-IDEX would be
preferentially reflecting lower levels of CHRM1, except for the
striatum, where CHRM1 levels are lower and the binding of theradioligand would reflect levels of CHRM1 and CHRM4. Althoughboth studies support lower levels of CHRM1/CHRM4 in people
with schizophrenia, neither of the studies were sufficiently powered
to identify the MRDS subgroup identified using postmortem
CNS (Scarr et al., 2009). Moreover, neither study quantified
CHRM1/CHRM4 in the hippocampus, a region important inmodulating learning and memory which have shown impairmentsin people with schizophrenia (Lodge and Grace, 2011).
More recent research has attempted to address some of thelimitations of the early studies on CHRMs in schizophrenia
by measuring CHRM1/CHRM4 using SPECT and 123 I-IDEX
binding in 30 medication-free people who were diagnosed witha psychotic disorder and were in the early phase of the disease(Bakker et al., 2018). In this study there was no evidence for the
subgroup of people with extremely low levels of CHRM1/CHRM4
that defined MRDS (Scarr et al., 2009). This could be because
the study included people with a range of psychotic disorders,which raises the possibility that MRDS may be specific toschizophrenia and would therefore be less apparent in a mixed
group. Importantly, this study did show a significant relationshipbetween lower 123 I-IDEX binding in the DLPFC and more severe
negative symptoms, supporting a more prominent role for the
CHRM1 in the genesis of those symptoms. This finding is inline with improvements in negative symptoms in a subset ofpeople after treatment with clozapine which were suggested to be
due to the clozapine metabolite n-desmethylclozapine acting as a
CHRM1 agonist (Meltzer, 1992, 1997; Costa-Dookhan et al., 2021).
Despite these encouraging results no data suggests that treatment
with n-desmethylclozapine as a monotherapy is beneficial in thetreatment of schizophrenia (Mendoza and Lindenmayer, 2009).
Another finding from the study of 123 I-IDEX to
CHRM1/CHRM4 binding in psychosis was that lower levelsof binding in the DLPFC was related to worse verbal learning andmemory scores whereas lower hippocampal binding predicted
worse delayed recall (Bakker et al., 2018). These findings were
further investigated in the same patients using functional magnetic
resonance imaging and a task measuring functional activation
during visual spatial learning and memory after treatment withbiperiden (Bakker et al., 2020). Importantly, biperiden is a selective
CHRM1/CHRM4 antagonist (10-fold more selective for CHRM1
over CHRM4) that is known to induce significant deficits inlearning and memory in both patients with schizophrenia andcontrols (Vingerhoets et al., 2017). The outcome of the study
with biperiden was the demonstration of a significantly greater
activation in response to biperiden in the parahippocampal andsuperior temporal gyrus in the people with psychosis during
both learning and memory tasks compared to controls (Bakker
et al., 2020). In addition, lower CHRM1/CHRM4 binding in the
hippocampus during learning and CHRM1 binding in the DLPFC
during memory, predicted a greater magnitude of abnormal
hyperactivity (Bakker et al., 2020). These data argue that there may
be a loss of CHRM1/CHRM4 reserve in people with psychotic
disorders early in the onset of the disorder that is giving an
increased sensitivity to biperiden.
Taken together, data from postmortem CNS show there
are lower levels of CHRM1/CHRM4 in some people withby a lack of highly selective radioligands and because suchstudies would preferably involve medication-free people with thedisorder. However, given the difficulties in studying CHRMs using
medication-free people with schizophrenia it has been deemedworthwhile to study people with schizophrenia who are not being
treated with anticholinergic drugs to control extrapyramidal-side
effects (Caradoc-Davies et al., 1986) or with atypical antipsychotic
drugs, such as olanzapine, that occupy CHRMs (Bymaster et al.,2003).
Molecular imaging studies measuring CHRMs in people
with schizophrenia have been possible using two non-selective
single photon emission compute tomography (SPECT) tracers 3quinuclidinyl-4-[123 I] iodobenzilate (123 I-QNB) and N-methyl-4[123 I] iododexetimide (123 I-IDEX) that have high affinities for both
the CHRM1 and CHRM4 (Muller-Gartner et al., 1992; Boundy
et al., 1995; Piggott et al., 2002). The distribution of both 123 IIQNB and 123 I-IDEX reflect the distribution of CHRM1 andCHRM4 in the human CNS showing high levels of uptake in thecortex, hippocampus, and striatum (Figure 1). Importantly, bothtracers are antagonists and therefore their signal is not specific tofunctional receptors in the high affinity state. The pharmacologicalcharacterization of 123 I-IDEX and 123 I-IQNB showed 123 I-IDEX
was a more selective CHRM1/CHRM4 tracer that has a higher
affinity for CHRM1 (picomolar range) over CHRM4 (nanomolar
range) and was preferentially displaced by CHRM1 agonists andantagonists (Bakker et al., 2015). Recognizing the limitationsof current ligands, there are ongoing efforts to identify agonist
positron emission tomography (PET) tracers that are able todifferentiate between CHRM1 and CHRM4 (Table 2; Ozenil et al.,2021). In addition, a fluorinated PET version of dexetimide has
been synthesized (Rowe et al., 2021) which has been reported to besafe to use in humans and has a CNS distribution consistent withthe ligand targeting CHRM1 and CHRM4.
The need for neuroimaging ligands has become critical to
study CHRM target engagement because of recently reported
clinical trials in people with schizophrenia. These trials included
phase II and III trials showing the efficacy of KarXT, which ispredicted to having its therapeutic benefits from the inclusion of
CHRM1/CHRM4 dual agonism, against the positive and negative
symptoms of schizophrenia (Brannan et al., 2021). In addition, therecent report that emraclidine, a specific CHRM4 PAM, lessons
psychotic and negative symptoms in people with schizophrenia(Krystal et al., 2022).
The initial neuroimaging study of CHRMs in schizophrenia
used 123 I-IQNB SPECT to show there were lower levels of
CHRM1/CHRM4 (30% in the striatum and 20% in frontal andtemporal regions) in the CNS of drug-free people with thedisorder compared to matched control subjects (Raedler et al.,2003). Notably, lower levels of 123 I-IQNB binding were associatedwith worse positive symptom severity. This study added to an
earlier study using 123 I-IDEX SPECT that reported lower levels of
CHRM1/CHRM4 in people with schizophrenia who were being
treated with risperidone (Lavalaye et al., 2001). Importantly, incontrast to some other atypical antipsychotics, risperidone does not
occupy CHRMs and therefore would not affect 123 I-IDEX binding(Lavalaye et al., 2001; Corena-Mcleod, 2015). In this study, thosewith the schizophrenia had approximately a 2.5-fold lower levelof CHRM1/CHRM4 in the frontal, striatal, temporal, and occipitalcortex compared to controls (Lavalaye et al., 2001). In interpreting

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FIGURE 1

An example of an image, in cool-warm colors, obtained after 123 I-IDEX SPECT scan registered and overlain onto T1 weighted anatomical MRI scanshowing high specific preferential binding to CHRM1 and CHRM4 receptors in the cortex, including the dorsolateral prefrontal cortex (DLPFC),
hippocampus, and striatum, with no uptake in the cerebellum, which is devoid of CHRM1 and CHRM4.

TABLE 2 Overview of radioligands used to evaluate M1 and M4 receptor binding in schizophrenia and promising selective positron emission
tomography (PET) radioligands for future research to understand functional changes in M1 and M4 receptors.

[123 ] I-IQNB[123 ] I-IDEX[11 ]C-LSN31721765[11 ]C-MK-6884

Imaging modality

SPECT

SPECT

PET

PET

Spacial resolution

Low

Low

High

High

Mode of action

Antagonist

Antagonist

Agonist (bitopic ligand)Positive allosteric modulator

Specificity

Non-selective CHRM1/M4

Non-selective CHRM1 > CHRM4

CHRM1 selective

CHRM4 selective

Signal interpretation

CHRM1 + CHRM4

CHRM1 + CHRM4 but CHRM1 in CHRM1
CHRM1 rich CNS regions

Basal binding can reflect basalcholinergic tone.
Simple tasks can be used to activate
areas of interest to reflect CHRM4
function

Reflect high affinity
state/g-coupled state

No

No

Yes

Yes

Published data

Schizophrenia
neurodegenerative disorders

Psychotic disorders
neurodegenerative disorders

Healthy volunteers only

Alzheimer’s disease

Radioligand
Radioligand
structure

Scarr et al., 2015; Fillman et al., 2016; Lai et al., 2016a; Dean et al.,2022) and the peripheral effects of xanomeline (Shekhar et al.,2008) was a problem in taking the drug into the clinic. Thus,
although the major focus on understanding schizophrenia has beenon the perturbations in CNS function and potential mechanismsthat could underlie the genesis of the constellation of symptoms
needed to make a diagnoses (American Psychiatric Association,2013) it is now argued that understanding peripheral biochemical
homeostasis will aid in developing diagnostic and theranostic
tools to aid in disease management (Do, 2023). Moreover it ispossible that breakdown in peripheral biochemical homeostasis
could be why people with schizophrenia experience other medical
conditions at a higher rate than in people without a psychiatricschizophrenia. Given the lack of agonist SPECT or PET tracers,
current studies provide no direct evidence as to whether there
is a loss of receptors in the high affinity (g-coupled) state inpeople with schizophrenia, but this limitation may be overcome
with the development of 11C-MK6884 (CHRM4 PAM) and 11C-LSN3172176 (bitopic CHRM1 agonist) PET tracers.

2.10. Health comorbidities andschizophrenia
There are now data that argue that the molecular pathology
of schizophrenia extends beyond the CNS (Glatt et al., 2005;

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FIGURE 2

A schematic showing the connections between the CHRM1 and CHRM4 and the symptoms associated with schizophrenia. The red boxes showconnections where there is a direct connection between and animal model and a symptom of schizophrenia. Brown boxes are symptom ofschizophrenia that can be connect to CHRM1 and CHRM4 through an intermediate finding, shown in green boxes, using animal models ormolecular studies. The absence of links to the negative symptoms reflects the absence of validated animal models and a poor understanding of thebiological processes generating these symptoms.

case of chronic obstructive pulmonary disease, it is significant
that the CHRM1−/− mouse has changes in bronchoconstriction(Struckmann et al., 2003) and a pan-CHRM1, 2 and 3 antagonist
is used to treat asthma (Matera et al., 2020). In addition, it has
recently been reported that the CHRM4 in the pedunculopontine
tegmental nucleus is a mediator of respiration in the rat (Lima et al.,2019). Finally, and somewhat surprisingly, the CHRM1−/− mousehas developmental abnormalities in the auditory cortex (Zhang
et al., 2005) which raises the intriguing possibility that a deficit
in CHRM1 may be involved in the increase in hearing loss inpeople with schizophrenia. Whilst all these observations currently
remain a matter of conjecture, the possibility of abnormal CHRM
signaling being involved in more than just the core symptoms ofschizophrenia could be worthy of investigation.

condition (Smith et al., 2013). Medical conditions that have a higher
occurrence in people with schizophrenia include viral hepatitis,
epilepsy, dyspepsia, liver disease, diabetes, blindness, thyroid
disorders, coronary heart disease, pain, psoriasis and eczema,
chronic obstructive pulmonary disease, migraine and hearing loss.
It has been suggested that people with schizophrenia may haveproblems with untreated medical conditions due to poorer accessto medical services (Carney et al., 2006) or due to unwanted side
effects of antipsychotic drug treatment (Mazereel et al., 2020).
However, with the recognition that the breakdown of biochemical
homeostasis in people with schizophrenia extends beyond the CNS(Scarr et al., 2015; Lai et al., 2016a) some consideration should
be given as to whether CHRM1 / CHRM4-mediated biochemical
abnormalities in people with the disorder could extend beyond the
CNS.
In considering potential roles of the CHRM1 and CHRM4 incomorbid central and peripheral disorders, it is significant that
mutations in the CHRM1 may be involved in the genesis of epilepsy(Marcé-Grau et al., 2021). In relation to the systemic illnesses
more prevalent in people with schizophrenia, recent reports of thepresence of CHRM1 on the pancreas (Pacher et al., 2019) and asa suggested drug target for the treatment of gastrointestinal andliver diseases (Tolaymat et al., 2021) are of interest. With regards topain and schizophrenia, it is interesting that xanomeline has beenshown to be an analgesic in the rodent models of inflammatory
and neuropathic pain (Martino et al., 2011). In the case of higher
rates of psoriasis and eczema, it is notable that the CHRM4 has
been shown to have a role in modulating skin keratinocyte adhesion(Nguyen et al., 2004) which is important in skin repair. In the

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3. Concluding remarks
The successful transition of the drug KarXT, a coformulation
containing the CHRM1/CHRM4 agonist, through a large phase2 trial (Brannan et al., 2021) and a phase 3 trial raises theprospect of a new treatment for schizophrenia that is not a
dopamine D2 receptor antagonist that may also have some partial
agonist properties. Moreover, early data from the trial of treating
schizophrenia with emraclidine indicate selectively targeting theCHRM4 may also be a mechanism to reduce psychotic and negative
symptom severity (Krystal et al., 2022). In addition, there areencouraging data showing the beneficial effects of using trace10

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Conflict of interest

amine-associated receptor 1 (TAAR1) agonists to treat thesymptoms of schizophrenia (Nair et al., 2022). It would therefore
seem possible that drugs with different mechanisms of actions
will be available for the treatment of schizophrenia and otherdisorders with similar symptoms. The completion of ongoing trials
will hopefully provide more information on clinical benefits andside-effect profiles to help clinicians decide on more personalized
treatment management plans.
Importantly, the discovery of the antipsychotic properties ofchlorpromazine precede understanding the critical role of its action
at the dopamine D2 receptor being critical to its antipsychotic
effects (Shen, 1999). This contrasts to efforts to develop drugs
to treat the symptoms of schizophrenia by targeting CHRMs
because, as outlined in this review, evidence from different linesof research have helped explain why such an approach would havetherapeutic benefits (Figure 2). Hence, if drugs targeting CHRMs
to treat schizophrenia do enter clinical use this will not only be an
exciting development for treating the disorder but would also argue
for more intensive studies of its molecular pathology to identify
potential new opportunities to develop new drug treatments. Suchdrugs will likely be necessary because, like in other areas of
medicine, it is unlikely one drug will provide an optimum therapy
for everyone within the syndrome of schizophrenia.

HU was a co-inventor of CUBIC and ACCEL and a co-founderof CUBICStars Inc., and ACCELStars Inc.; neither company has afocus on muscarinic receptor research. AB was employed by Sosei
Heptares and GB was an external consultant contracted by Sosei
Heptares. Whilst Sosei Heptares is a company developing drugs
targeting muscarinic receptors, they played no role in funding thiswork other than salary paid as employer and for contracted work.
No financial support was given to aid in the publication of thismanuscript by any of the employing institutions and the contentsof the review reflects the consensus view of the authors.
The remaining authors declare that the research was conductedin the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.

Publisher’s noteAll claims expressed in this article are solely those of theauthors and do not necessarily represent those of their affiliatedorganizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

Author contributions
BD prepared portions of the review and integrated individual
contributions into a final version for review. GB and HU
prepared specific sections of the review based on their specialist
knowledge. RK provided the specialist clinical oversight. All
authors contributed to the conceptualization of the scope of thisreview and involved in preparing the final manuscript submitted
for publication.

Supplementary material
The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fncel.2023.
1124333/full#supplementary-material

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