Abstractl
Background/Objectives
Studies have shown widespread alterations in different components of the cholinergic system in schizophrenia, but to date the evidence has not been systematically reviewed and summarized. Here, we systematically review imaging and post-mortem studies on the central cholinergic system in schizophrenia/schizoaffective disorder.
Subjects/Methods
Searches were performed in Embase and Medline. Study designs included cross-sectional case control studies comparing individuals with schizophrenia/schizoaffective disorder to control population. Risk of bias was assessed with the NIH/NHLBI tool for Quality Assessment of Case-Control Studies. The current study followed the PRISMA 2020 guidelines (PROSPERO: CRD42023402126).
Results
A total of 3259 studies were screened and 61 met eligibility criteria for the systematic review, including 8 in vivo neuroimaging and 53 post-mortem studies. About 74% of these studies described significant alterations, most often reductions in either muscarinic or nicotinic receptor levels in schizophrenia. We also conducted 3 meta-analyses showing reductions in M1/M4 muscarinic receptors in the striatum (g = −0.809, k = 3, n = 108), hippocampus (g = −0.872, k = 3, n = 84), and fronto-cingulate cortex (g = −0.438, k = 4, n = 295). Six neuroimaging studies reported associations with clinical symptom severity measures, and four investigations with cognitive dysfunction.
Conclusions
Our review demonstrates a widespread decrease in muscarinic and nicotinic receptor levels in schizophrenia, evident in both neuroimaging and post-mortem studies. Our meta-analyses show large to moderate effects for the reductions in M1/M4 muscarinic receptors in the striatum, hippocampus, and fronto-cingulate cortex. Limitations and future directions for the field are discussed.
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Introduction
Since the mid-20th century, researchers have hypothesized that the cholinergic system could be targeted for the treatment of schizophrenia [1]. Observations that anticholinergic drugs could produce psychotic symptoms further suggested that abnormal cholinergic function might underly the pathophysiology of schizophrenia [2, 3]. A growing body of literature using neuroimaging or post-mortem samples now shows changes in cholinergic neurotransmission in this disorder, and multiple cholinergic agents are being developed and tested for the treatment of schizophrenia [4,5,6,7,8,9].
Cholinergic neurotransmission involves acetylcholine release and complex pre- and post-synaptic molecular apparatus (see Fig. 1). The cholinergic system is essential for synaptic plasticity, modulation of excitation-inhibition balance and oscillations, as well as the coordination of functional networks [10, 11]. This system modulates a wide variety of neurobehavioral functions from sensory processing and perception to cognition, memory, emotional regulation, and motivation [12,13,14,15,16]. Since disruptions of these functions are thought to manifest as symptoms of schizophrenia, abnormal cholinergic signaling may partly underly the clinical presentation of schizophrenia [17,18,19,20,21]. Moreover, the cholinergic system modulates dopamine release in the striatum, where the cholinergic receptors are localized on most GABAergic interneurons as well as dopamine and glutamate terminals [22,23,24,25,26]. Specifically, activation of the M1 and M4 receptors dampens striatal dopamine release whereas the activity of various nicotinic receptor subtypes facilitate dopamine release in this brain region [27,28,29]. Consequently, some of the effects of abnormal cholinergic signaling in schizophrenia may be mediated via modulation of dopamine release [18,19,20].
Acetylcholine is synthesized from choline and acetyl-coA by choline acetyltransferase, packed into vesicles by a vesicular acetylcholine transporter, and, after release, broken down by acetylcholinesterase. The effects of released acetylcholine are conveyed by nicotinic ligand-gated ion channels and muscarinic metabotropic (G-protein coupled) receptors. There are 5 muscarinic receptor subtypes: M1–M5. Muscarinic M2 receptors can act as pre-synaptic auto-receptors on cholinergic neurons, while the other subtypes act mainly as heteroreceptors on other cholinoceptive neuronal types (both pre- and post-synaptically). Nicotinic receptor subunits include α3, α4, α5, α6, α7, β2, and β4. The most common nicotinic receptors in the brain are the heteromeric α4β2 and the penta-homomeric α7 receptors, which are located both on cholinergic and cholinoceptive neurons (e.g., pre- and post-synaptically on dopamine neurons). Cortical and hippocampal innervation is primarily provided by projections from the basal forebrain and brainstem cholinergic nuclei, while the majority of acetylcholine release in the striatum is accounted for by cholinergic interneurons. Some evidence suggests that cholinergic neurotransmission also occurs by slow volume transmission across extracellular space, in addition to the classical synaptic transmission. Image created in BioRender.com.
In addition to post-mortem and imaging studies, the involvement of the cholinergic system in schizophrenia is also supported by pharmacological studies. Anticholinergic agents can induce psychotic-like experiences and cognitive deficits in healthy controls [30]. In schizophrenia, anticholinergic activity can exacerbate psychotic symptoms as well as worsen cognitive, brain structural, and functional impairments [30,31,32]. Most prominently, many recent clinical trials of cholinergic agents have shown promising results in the treatment of this disorder [8, 9]. Specifically, the brain-penetrant muscarinic (M1/M4) agonist xanomeline (combined with the peripheral blocker trospium) has demonstrated efficacy in improving positive, negative, and cognitive symptoms of schizophrenia [8, 33, 34]. These pharmacological findings lend additional credibility for the postulated cholinergic involvement in this mental health disorder.
Given recent developments in cholinergic therapeutic agents and increased interest in this system in schizophrenia, it is important to systematically summarize the existing evidence for the involvement of the cholinergic system in the pathophysiology of schizophrenia. Changes in the cholinergic system in schizophrenia have been studied in post-mortem samples as well as in vivo using positron emission tomography (PET) and single photon emission computed tomography (SPECT) [4, 5, 35]. There are multiple narrative reviews describing cholinergic alterations in schizophrenia [33, 36,37,38]. However, the human experimental data on the topic has never been systematically reviewed. Therefore, we conducted a systematic review (and meta-analyses when feasible) to identify, summarize, and combine the broad literature quantifying neuronal cholinergic alterations in the brain of individuals with schizophrenia.
Methods
To better understand the potential alterations associated with schizophrenia, we systematically reviewed the human empirical data on the brain cholinergic system in this disorder relative to controls. The systematic review process used in the current protocol follows the PRISMA 2020 guidelines (see supplementary material) and was registered in PROSPERO in March 2023 (https://www.crd.york.ac.uk/prospero/display_record.php?ID = CRD42023402126) [39].
Search strategy
Systematic searches were performed in Ovid Embase and Medline (see Supplementary Table S1 for example of Ovid Embase search strategy; last search: June 2024).
Inclusion and exclusion criteria
Sources needed to include original, peer-reviewed data where direct experimental measures of the cholinergic system in the human brain (e.g., cholinergic receptors, transporters, enzymes (protein or mRNA expression levels), or neuronal counts) were compared between a sample of persons with schizophrenia or schizoaffective disorder and a control sample. The diagnosis of schizophrenia or schizoaffective disorder needed to be confirmed using a validated diagnostic assessment tool that adheres to either the Diagnostic and Statistical Manual of Mental Disorders’ (DSM-III or higher; American Psychiatric Association) or the International Classification of Diseases’ (ICD-9 or higher; World Health Organization) diagnostic criteria. Included study designs encompassed cross-sectional, case control, and cohort studies (with control groups). Control trials studies were only eligible if they reported baseline measures of cholinergic markers in comparison to a control group. Sources were limited to the English language. Sources were ineligible if they included purely DNA sequencing without measuring cholinergic expression markers; measured the cholinergic system outside of the brain (e.g., blood cells) or in non-neuronal cells; or utilized animal models. Reviews, meta-analyses, and conference presentations were not eligible.
Data extraction
All collected sources were uploaded into Covidence (https://www.covidence.org), an online software that facilitates the systematic review process. Two members of the research team reviewed and screened the titles and abstracts of the collected sources according to the predefined inclusion and exclusion criteria (ZS & RH). The remaining sources underwent full-text review/screening by two members according to the predefined criteria (ZS & JM). Data on general study characteristics, methodology, sample composition, and relevant quantitative results were extracted from the included sources and recorded in a customized data extraction spreadsheet independently by two of three members of the team (ZS, RA & JM). At each step, disagreements of relevant data were resolved through discussion by two members of the research team, until consensus was reached.
Risk of bias assessment
The quality of eligible sources was determined using the NIH/NHLBI tools for Quality Assessment of Case-Control Studies (https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools). Two reviewers independently assessed the quality of each included source (ZS, RA, JM). Disagreements were resolved through discussion, until consensus was reached.
Data synthesis
We performed a narrative synthesis of the findings. General data synthesis described the following characteristics: study characteristics (i.e., author names, year, study type), smoking status, medications, sample/group characteristics (i.e., specific diagnosis, sample size, demographics), component of the cholinergic system quantified (including relevant methodological information, such as ligand/probe, etc.), and study results (i.e., between-group differences in cholinergic system components). Results were tabulated according to these measures.
We also stratified the results according to the study type, methodology, cholinergic components assessed (e.g., receptors, transporters, enzymes, mRNA expression, and neuronal counts) and the brain regions assessed. We reported findings narratively and conducted a meta-analysis when ≥ 3 studies showing effect sizes per methodological assay/measure were available for a given brain region.
Meta-analytical statistics
When 3 or more studies using the same cholinergic probe/assay in the same brain region were identified, meta-analyses were performed using the metafor package in R with random effect modeling, unbiased estimator, and Hedge’s g statistics, which is more robust with small sample sizes [40]. R code is available upon request. The Q-Statistic was used to test heterogeneity and publication bias was visually evaluated with funnel plots.
Results
A total of 61 studies met eligibility criteria for the current systematic review, including eight in vivo neuroimaging studies and 53 post-mortem investigations (see Supplementary Fig. S1 for PRISMA diagram). Of the total 61 included investigations, 45 showed alterations in the cholinergic system in schizophrenia (see Table 1 for details).
In vivo neuroimaging studies
There are eight neuroimaging studies on the cholinergic system in schizophrenia: one study used [123I]IQNB, a non-selective muscarinic receptor antagonist; two studies used [123I]5-IA-85380, a nicotinic β2* agonist; three studies used [18F]-ASEM, a nicotinic α7 receptor antagonist; one study used 2-[18F]FA, an α4β2 agonist; and one study used [18F]VAT, a vesicular acetylcholine transporter inhibitor [4, 7, 35, 41,42,43,44,45]. See Fig. 2a for a visual summary of neuroimaging findings in schizophrenia.
Each panel displays a visual summary in the form of brain maps with corresponding tables, highlighting the main findings from the systematic review [55, 56]. Panel a) shows in vivo neuroimaging evidence. Panels b) and d) display findings on post-mortem muscarinic receptors and mRNA, respectively. Panel c) highlights findings on post-mortem nicotinic receptors as well as mRNA. k = #: # of studies represented in the panel. ↑ Higher in schizophrenia relative to controls, ↓ Lower in schizophrenia relative to controls, ~ No group difference, 6–46 Brodmann Areas #, FC Frontal Cortex, PC Parietal Cortex, CC Cingulate Cortex, ACC Anterior Cingulate Cortex, PCC Posterior Cingulate Cortex, TC Temporal cortex, STG Superior Temporal Gyrus, OC Occipital Cortex, Cau Caudate, Put Putamen, Str Striatum (caudate/putamen), Tha Thalamus, Hip Hippocampus, Ms Muscarinic receptors (combined subtypes), M# Muscarinic receptor subtype #, Ns Nicotinic receptors (combined subtypes), α# α nicotinic receptor subunit/subtype #, β2 β2 nicotinic receptor subunits, vAChT Vesicular Acetylcholine Transporters. *Confounds, including a large smoking and age mismatch between groups, render findings inconclusive.
Six of the seven studies on muscarinic or nicotinic receptors (density or availability thereof) found reductions in schizophrenia relative to controls [4, 7, 35, 43,44,45]. One study compared older patients with schizophrenia who smoked to younger healthy controls who mostly did not smoke, thus not allowing to conclude on effect of illness [42]. The report on vesicular acetylcholine transporters did not find evidence for a group difference [41]. The small number of in vivo studies, their use of a variety of radioligands, regions-of-interest, or shared participants (follow-up reports of the same study) precluded us from conducting a meta-analysis for the neuroimaging investigations.
Associations with symptoms of schizophrenia
Six neuroimaging studies also reported significant associations between lower cholinergic receptor levels and more severe clinical or cognitive symptoms, while one of the investigations on receptors did not report this type of analysis [4, 7, 35, 42,43,44,45]. Higher levels of positive symptoms were associated with lower levels of muscarinic receptors in the frontal cortex as well as caudate/putamen [4]. Similarly, higher levels of positive symptoms correlated with lower levels of α7 nicotinic receptor in widespread cortical and subcortical regions in individuals with recent-onset psychosis (schizophrenia spectrum and bipolar I disorder) [43]. A negative correlation was also found between negative symptoms and β2* nicotinic receptor availability in the striatum, frontal, and parietal cortex [7]. Additionally, a negative correlation was shown between β2* nicotinic receptor availability in frontal and parietal cortices and cognitive deficits in smokers with schizophrenia [7]. Similarly, α7 nicotinic receptor availability negatively correlated with cognitive deficits in the temporal, occipital and cingulate cortices as well as the striatum and hippocampus, in recent-onset psychosis [35, 43]. Lastly, the study assessing vesicular acetylcholine transporters, although not showing group differences, found higher transporter levels in many brain cortical and subcortical regions to correlate with higher positive symptoms and with lower cognitive ability in individuals with schizophrenia [41].
Post-mortem studies
Constituents of the cholinergic system in post-mortem samples have been quantified using autoradiography, in situ hybridization, Western blots, enzyme assays, and PCR [5, 46,47,48,49]. Of the 53 eligible post-mortem studies, 30 measured muscarinic receptor proteins, mRNA, or the coupling of receptors with intracellular targets; 17 assessed nicotinic receptor protein and/or mRNA; five reported directly on neuronal cholinergic/cholinoceptive populations; and four investigated cholinergic enzymes. Notably, three studies assessed cholinergic receptors along with either enzymes or neuronal populations. M1/M4 receptors were the most widely studied target using [3H]pirenzepine. Other commonly studied targets were the nicotinic α7 and muscarinic M2/M4 receptors, using [125I]α-bungarotoxin and [3H]AF-DX 384, respectively. In terms of brain regions, a majority of studies have focused on the frontal and cingulate cortices, striatum, and hippocampus.
Muscarinic receptors
A total of 30 studies investigated muscarinic receptor expression, protein, or coupling measures. Twenty reported decreases in the post-mortem brain in schizophrenia compared to controls, whereas three investigations showed bidirectional changes, only two evidenced an increase, and five studies found no group differences. Figure 2b and d provide visual representations of muscarinic receptor and mRNA findings, respectively. The most used cholinergic probe in post-mortem schizophrenia literature is [3H]pirenzepine, a pseudo-selective muscarinic antagonist. This tracer has the highest affinity for the muscarinic M1 receptor, followed by the muscarinic M4 receptor [50]. In post-mortem human cortex, where the ratio of M1 to M4 receptors is also relatively higher, the majority of [3H]pirenzepine binding is attributable to M1 receptors (≳80%) [38, 51]. Whereas M4 receptors are relatively more abundant in the striatum, resulting in a more mixed [3H]pirenzepine signal to which both M1 and M4 receptor levels contribute [38, 52].
Meta-analytical findings
There was a sufficient number of independent investigations assessing [3H]pirenzepine binding in the striatum (k = 3, n = 108), hippocampus (k = 3, n = 84), and the fronto-cingulate cortex (k = 4, n = 295) to conduct meta-analyses. Random effect models yielded large effect sizes in the striatum (g = −0.809 [CI: −1.492, −0.125], p < 0.05) and the hippocampus (g = −0.872 [CI: −1.326, −0.418], p < 0.001), and a medium effect size in the fronto-cingulate cortex (g = −0.438 [CI: −0.670, −0.206], p < 0.001), in schizophrenia relative to controls. See Fig. 3 for forest plots and effect size contributions.
Forest plots of Random Effect (RE) meta-analyses of [3H]pirenzepine binding assays, representing M1/M4 receptor density, in the post-mortem (a) striatum, (b) hippocampus, and (c) fronto-cingulate cortex of individuals with schizophrenia relative to controls. Percent contribution to pooled effect size (Hedge’s g) shown as a square. Whiskers show confidence intervals (95%). For the meta-analysis in fronto-cingulate cortex (c), Zavitsanou and colleagues (2004) assessed anterior cingulate region and the other three studies assessed Brodmann Area 9.
Due to the low number of studies included in each meta-analysis (k = 3–4), publication bias was only assessed visually with funnel plots. The funnel plots from our meta-analyses did not highlight significant asymmetry beyond what could be expected from the random distribution of three unbiased publications (see Supplementary Fig. S2). Furthermore, no significant heterogeneity in effect sizes across studies included were found, suggesting the investigations are assessing the same underlying effect and variations in effect sizes could be attributed to sampling error rather than differences in study populations (striatum: Q(df = 2) = 4.67 (p = 0.10); hippocampus: Q(df = 2) = 2.00 (p = 0.37); and fronto-cingulate cortex: Q(df = 3) = 0.92 (p = 0.82)).
Nicotinic receptors
There were 17 studies on nicotinic receptors, and 12 of them found changes in schizophrenia. Changes in nicotinic receptor subtypes in post-mortem schizophrenia samples were less unidirectional than those of muscarinic receptors, with certain subtypes downregulated in a region but upregulated in another (see Table 1). For instance, α4β2 receptor levels may be decreased in the hippocampus, whereas the same receptor may be increased in the cingulate cortex [53, 54]. See Fig. 2c for a visual summary of nicotinic receptor and mRNA findings in schizophrenia [55, 56].
Cholinergic enzymes and neurons
Four studies investigated cholinergic enzymes (e.g., choline acetyltransferase and acetylcholinesterase) in the post-mortem brain of individuals with schizophrenia [48, 57,58,59]. Only one study reported increased choline acetyltransferase in the occipital cortex and angular gyrus of patients with schizophrenia relative to controls [57]. The remaining three studies did not find group differences.
Four of the five eligible histological studies found reductions in cholinergic or cholinoceptive neuron/cell populations in schizophrenia relative to controls, in the striatum and specific subregions of frontal and occipital cortices [47, 48, 58, 59].
Risk of bias
The majority of studies included in our review were rated as fair in design quality and risk of bias assessments (see Supplementary Table S2) because they did not statistically control for potential confounds (e.g., smoking, medications) in their analyses. Many also did not include sample size justification or power analysis. This may reflect the inherent limitations of the field that we reviewed. Sample sizes in post-mortem and nuclear imaging studies are often limited by feasibility, which in turn, limits the statistical power to account for confounds.
Discussion
Cholinergic alterations in schizophrenia
We systematically reviewed and conducted meta-analyses to summarize the evidence of alterations to the cholinergic system in the brain of individuals with schizophrenia. Our findings establish that multiple components of the cholinergic system across several cortical and subcortical brain regions are affected in schizophrenia. Evidence from in vivo neuroimaging studies demonstrate significant regional decreases in cholinergic receptor availabilities (i.e., muscarinic receptors as well as nicotinic α7 and β2* receptors). Likewise, the majority of reported changes in post-mortem schizophrenia samples are decreases in markers of the cholinergic system, with few exceptions.
The most studied regions in post-mortem studies were the striatum, hippocampus, as well as the frontal and cingulate cortices. We conducted meta-analyses of M1/M4 muscarinic receptor densities in these regions and found a significant decrease with large effect sizes in the striatum (g = −0.809) and hippocampus (g = −0.872), as well as a medium effect size in the fronto-cingulate cortex (g = −0.438). Importantly, the small number of studies included in each meta-analysis (k = 3–4) is a limitation and warrants further research. These findings remain of particular interest considering the efficacy of agents targeting muscarinic receptors in reducing symptoms of schizophrenia [8, 9, 60]. Both xanomeline (M1/M4 agonist) and emraclidine (M4 positive allosteric modulator) have shown significant therapeutic effects in human clinical trials [8, 9]. Furthermore, it is noteworthy to mention that many studies included in this review used pseudo-selective probes and assays [51, 53, 61, 62]. Specifically, our meta-analyses rely on [3H]pirenzepine binding assays that do not allow complete separation of signal from M1 and M4 receptors [61, 63]. Regarding our findings in the fronto-cingulate cortex, other lines of evidence suggest that only M1 levels would be altered. In Brodmann Area 9, one of the regions included in that meta-analysis, Western Blots and PCR experiments have shown a decrease in M1 receptors and mRNA, but unchanged M4 receptor levels [5]. As for the findings in the striatum and hippocampus, lower [3H]pirenzepine binding could reflect lower M4 levels because M1 mRNA expression was found to be unchanged in those brain structures [63, 64]. Consistent with this, there is evidence that M4 mRNA levels are decreased in the hippocampus, whereas data on the striatum are lacking [64]. However, the attribution of [3H]pirenzepine signal remains speculative in those brain regions, since mRNA and protein levels do not always correspond [65]. In the future, it may be feasible to quantify individual muscarinic receptor subtypes in vivo with selective radiotracers [66, 67].
Cholinergic system and symptoms of schizophrenia
The cholinergic system is crucial to multiple neuronal circuits that support neurobehavioral functions affected in schizophrenia (e.g., cognitive processes, sensory processing, motivation, and emotional regulation) [12,13,14,15,16]. The majority of in vivo studies we identified in this review show a link between symptoms of schizophrenia and alterations in the cholinergic system. On one hand, greater positive symptom severity has been found to correlate with lower muscarinic receptors, lower α7 nicotinic, as well as increased vesicular acetylcholine transporters [4, 41, 43]. On the other hand, greater negative symptom severity has been associated with less β2* nicotinic receptors as well as lower M1 muscarinic receptors [7, 45, 68]. Additionally, greater cognitive deficits may correlate with lower α7 nicotinic and M1 muscarinic receptors [43, 44, 68]. Greater cognitive impairments may also correlate with lower choline acetyltransferase activity and increased vesicular acetylcholine transporters [41, 48]. Although these associations need to be replicated in larger samples, they lend credibility to the cholinergic involvement in the pathophysiology of schizophrenia. The implication of the cholinergic system in schizophrenia is further supported by clinical trials showing promising therapeutic effects of novel cholinergic agents [8, 9, 34].
Changes in striatal cholinergic system
The striatum has the densest cholinergic innervation and is a crucial site of cholinergic influence on neurobehavioral functions, such as learning, attention, decision-making, emotional regulation, and motivation [18, 41, 69,70,71]. Our meta-analytical post-mortem results emphasize a large decrease in M1/M4 muscarinic receptors in the striatum, suggesting a breakdown of cholinergic modulation in this region in schizophrenia. These two muscarinic receptor subtypes localize to multiple neuronal populations in the striatum, but most prominently post-synaptically on medium spiny neurons [72,73,74,75]. Both M1 and M4 are essential to the balanced control of dopaminergic neurotransmission in the striatum, which is associated with clinical symptom severity in schizophrenia [27, 28, 76,77,78]. Hence, it is not surprising that striatal muscarinic receptor availability is negatively correlated with positive symptoms [4]. Results are less consistent for nicotinic receptors in the striatum in schizophrenia. Some evidence suggests total nicotinic receptors may be increased in this region, while combined levels of β2* subunits may be unchanged, and α7/α4β2 receptor subtypes may be decreased [43, 79, 80]. Interestingly, lower α7 nicotinic receptor availability in the striatum is also associated with greater positive symptoms [43]. Studies of pre-synaptic markers of cholinergic innervation in the striatum have yielded opposing results and more research is required to conclude on whether it may be altered in schizophrenia [41, 46, 47, 81].
Alterations in the cholinergic system in the frontal and cingulate cortices
Our meta-analysis demonstrated significant reductions in muscarinic M1/M4 receptors in the fronto-cingulate cortex (combining data from Brodmann Area 9 and anterior cingulate) in schizophrenia. The frontal and cingulate cortices are essential to cognition, decision-making, social behavior, attention, and self-referential thought, whose function is disrupted in schizophrenia [82,83,84,85,86,87]. In the frontal cortex, there is evidence for a decrease in the number of cortical neurons displaying M1 receptors in schizophrenia relative to controls, and impaired M1 signaling correlates with cognitive deficits and negative symptoms in vivo in psychosis [68, 88]. Total muscarinic receptor availability in the frontal cortex also correlates with positive symptoms of schizophrenia [4]. Overall, alterations in muscarinic receptors in schizophrenia show a subtype-specific pattern with some, but not all, subtype-specific mRNA findings being concordant with receptor alterations [5, 6, 49, 89,90,91,92]. The nicotinic α7 receptors, which modulate excitatory neurotransmission, may be decreased in frontal and cingulate cortices, and lower α7 receptor availability may also be linked to greater positive symptom severity [43, 93]. Pre-synaptic markers of cholinergic innervation seem unchanged in the fronto-cingulate cortex in schizophrenia, but higher vesicular acetylcholine transporters in that region may be correlated with increased positive symptoms and poorer working memory performance [41, 58]. In summary, the literature shows region-specific muscarinic M1 and nicotinic α7 receptors abnormality in the fronto-cingulate cortex and associations with schizophrenia symptoms.
Cholinergic changes in the hippocampus
Our review indicates that specific components of the cholinergic system may also be altered in the hippocampus, specifically a large decrease in M1 and/or M4 receptors [54, 61, 64]. M1 receptors are primarily located on soma and dendrites of pyramidal and granule cells, where they potentiate NMDA and AMPA receptor signaling, and exert essential procognitive effects [94,95,96,97,98]. Research suggests that M4 muscarinic receptors localize to GABAergic interneurons and glutamatergic fibers in the hippocampus, modulate hippocampal circuits, and are vital to cognitive function [94, 99,100,101]. Thus, a breakdown of M1 or M4 signaling in schizophrenia may contribute to related cognitive impairments [102].
Total nicotinic receptors in the hippocampus may also be lower in schizophrenia [54]. Conversely, in vivo data suggest reductions in hippocampal α7 nicotinic receptors, which is supported by post-mortem mRNA changes [43, 103, 104]. The α7 is the most expressed nicotinic subtype in the hippocampus, localizing ubiquitously pre- and post-synaptically on interneurons, pyramidal cells and projection terminals [105]. α7 nicotinic receptors can enhance GABA and glutamate release and play a crucial role in cognitive functioning, with association with cognitive deficits in first-episode psychosis [43, 106]. Interestingly, nicotine use may have a differential effect on the expression of the α7 receptors in schizophrenia relative to controls, suggesting aberrant regulation in schizophrenia specifically [7, 103]. There is also evidence for lower surface availability of α7 receptors suggestive of altered assembly or trafficking in schizophrenia [103].
Similarly to the striatum, it is unclear whether pre-synaptic markers of cholinergic innervation in the hippocampus are altered in schizophrenia due to conflicting results [41, 81, 107]. Nonetheless, there is evidence that higher levels of hippocampal vesicular acetylcholine transporters are associated with poorer working memory performance and increased positive symptoms [41]. In sum, hippocampal cholinergic activity may be dysregulated in schizophrenia, and diverse cholinergic alterations are plausibly contributing to cognitive deficits and positive symptoms associated with the disorder.
Confounding factors, limitations, and future directions
Some limitations have been identified in the current literature about the cholinergic system in schizophrenia and future directions are suggested in this review (see Table 2). Although muscarinic M1/M4 receptors are the most studied components of the cholinergic system in schizophrenia, there is only one study that assessed their levels separately with Western Blots [5]. There is a lack of studies on pre-synaptic cholinergic function in schizophrenia in general, and even less investigations assessing how pre- and post-synaptic markers might be related. Only one study has investigated pre- and post-synaptic cholinergic markers in the same sample. That study found unchanged choline acetyltransferase in both patients with and without a deficits in M1/M4 receptors, suggesting that at least some part of the pre-synaptic cholinergic system is intact in patients with post-synaptic deficits [59]. In the future, systematic investigations including both pre- and post-synaptic markers should be conducted to assess whether one could be related to the other. This would also serve as a preliminary step in determining whether a primary cholinergic deficit might be driving the other cholinergic changes seen in schizophrenia. The difficulty in distinguishing illness from medication effects in the current literature is complicated by the cholinergic affinities of many antipsychotics (e.g., clozapine) and the use of anticholinergics to treat related pyramidal side effects. Many regional cholinergic alterations seem to be significant for individuals with schizophrenia regardless of their medication, while others may only be found in patients treated with benztropine, an anticholinergic medication [108]. Additionally, schizophrenia patients may be particularly vulnerable to the adverse cognitive effects of anticholinergic drugs, perhaps because of a primary cholinergic deficit in this illness [109]. This points to the importance of careful consideration when using anticholinergic medications in this disorder [110]. Furthermore, current knowledge highlights that smoking is a risk factor for schizophrenia and a predictor of relapse of psychotic episodes, but how smoking affects the cholinergic system in schizophrenia needs to be further explored [111,112,113,114]. This is of particular importance given the high prevalence of smoking in schizophrenia [115]. Findings regarding nicotinic receptors especially must be interpreted with caution because smoking status was not available in many of the included studies and some data suggest that nicotine may affect gene expression and upregulate certain nicotinic subtypes [103].
The potential cholinergic contribution in the pathogenesis of schizophrenia is supported by correlations between cholinergic markers and clinical symptoms, as well as cholinergic involvement in the neurobehavioral functions known to be impaired in schizophrenia [4, 17,18,19,20,21, 43]. However, current study designs do not allow to ascertain if cholinergic alterations could underly some of the other neuronal changes seen in schizophrenia, such as increased dopamine synthesis. There are very little data in early course psychosis, clinical high-risk, or in relatives of individuals with schizophrenia. It is also unclear whether cholinergic disruptions could drive symptoms only in a subgroup of individuals with schizophrenia [116].
Conclusion
To the best of our knowledge, the present study represents the first systematic review and meta-analyses of the broad literature of human brain studies on cholinergic alterations in schizophrenia. Widespread cholinergic alterations are reported, especially reductions in M1/M4 muscarinic receptors in the striatum, hippocampus, and fronto-cingulate cortex. In vivo studies also suggest reductions in cholinergic receptors and associations with symptom severity. Whether these cholinergic alterations are central to the pathophysiology of schizophrenia or are secondary to the effects of medications and/or smoking needs to be clarified by further research. Despite these limitations, current evidence strongly supports the continued development of cholinergic agents for treating schizophrenia symptoms, both as primary antipsychotic medications and as adjuvants aiming at reducing more specific symptom clusters, such as cognitive deficits. Future research should focus on disentangling illness, medication, and smoking effects as well as investigating the pre-synaptic and post-synaptic cholinergic system across different stages of illness. The continued development of more specific in vivo radioligands is also needed and could be used to identify or confirm potential cholinergic biomarkers or subgroups with particular cholinergic impairments. The current review highlights the cholinergic system as a plausible contributor to the pathophysiology of schizophrenia as well as a promising target for novel treatments.
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Acknowledgements
The current systematic review was supported by the Canadian Institutes of Health Research (support provided to LT and SG; Grant #173227), the Emerging Research Innovators in Mental Health (eRIMh) Program (support provided to LT and SG), and the Vanier Canada Graduate Scholarship Program (scholarship provided to ZS; Award #455165).
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ZS: protocol development, search, abstract screening, full-text review, data extraction and risk of bias assessment, analyses, original draft, writing, figures and tables. JM: full-text review, data extraction, risk of bias assessment, figures. RA: data extraction and risk of bias assessment. RH: abstract screening, figures. MS: protocol development, reviewing, editing. MK: protocol development, reviewing, editing. LT: conceptualization, writing, reviewing, editing, analyses, supervision. SG: conceptualization, writing, reviewing, editing, supervision.
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MS received honoraria/has been a consultant for AbbVie, Boehringer-Ingelheim, Otsuka. SG received honoraria/has been a consultant for Boehringer-Ingelheim (Canada) Ltd. RH previously owned shares in Karuna Therapeutics and received two knowledge dissemination grants from Otsuka to organize local trainee conferences.
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Saint-Georges, Z., MacDonald, J., Al-Khalili, R. et al. Cholinergic system in schizophrenia: A systematic review and meta-analysis. Mol Psychiatry 30, 3301–3315 (2025). https://doi.org/10.1038/s41380-025-03023-y
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DOI: https://doi.org/10.1038/s41380-025-03023-y
