The mechanism underlying tardive dyskinesia: an exploratory test of the dopamine overactivity hypothesis using MRI imaging of midbrain neuromelanin

The mechanism underlying tardive dyskinesia: an exploratory test of the dopamine overactivity hypothesis using MRI imaging of midbrain neuromelanin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The mechanism underlying tardive dyskinesia: an exploratory test of the dopamine overactivity hypothesis using MRI imaging of midbrain neuromelanin Bernard R. Bukala, Luke J. Vano, Richard Carr, Alistair Cannon, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9245393/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rationale Tardive dyskinesia (TD) is a neurological syndrome of involuntary repetitive movements which results from treatment with antipsychotic medication. The pathoetiology of TD is not well understood but a possible mechanism involves dopaminergic overactivity in the nigrostriatal pathway. If this theory is correct, then levels of neuromelanin (a long-term marker of dopaminergic activity) should be higher in people with TD than those without TD. Objectives The aim of the study was to test the hypothesis that neuromelanin levels are higher in patients with TD relative to those without TD. Methods Data from 27 participants (TD: n = 13; non-TD n = 14) with a diagnosis of schizophrenia, all taking antipsychotic drugs, was used. Neuromelanin was measured in the midbrain via Magnetic Resonance Imaging (MRI) and compared between the groups. Movement symptoms were measured using the Abnormal Involuntary Movement Scale (AIMS), and antipsychotic dose recorded. Results MRI-measured neuromelanin levels were significantly higher in patients with TD, as compared to those without (mean = 0.223; t(17.27) = 3.817, p = 0.001; g = 1.386, 95% CI=[0.559, 2.213]). This remained significant after controlling for age, sex, substantia nigra/ventral tegmental area volume and antipsychotic dose (ANCOVA: F(1,20) = 12.08, p = 0.0024; adjusted β = 0.0301, 95% CI [0.0120, 0.0481]). The most pronounced difference was seen in the ventral substantia nigra. Conclusions The finding of higher midbrain neuromelanin in patients with TD aligns with the dopamine overactivity hypothesis of TD aetiology. It also supports a neurobiological basis for treatment of TD with drugs that target presynaptic dopamine activity, such as VMAT2 inhibitors. Additionally, it identifies the ventral substantia nigra as a key locus. Future longitudinal studies are needed to delineate if dopamine overactivity develops in response to antipsychotic treatment or is a trait vulnerability marker for risk of TD. Psychosis Neuroimaging Pharmacology Figures Figure 1 Figure 2 Introduction Tardive dyskinesia (TD) is a neurological syndrome characterised by involuntary, repetitive, non-rhythmic movements.(Salem et al., 2017 ) It commonly presents with orofacial symptoms, but can also involve limbs, digits, and truncal, pharyngeal or diaphragmatic muscles.(American Psychiatric Association, 2013 ) TD results from exposure to anti-dopaminergic drugs, most often antipsychotics, and symptoms usually emerge after at least a few months of treatment.(American Psychiatric Association, 2013 ; Salem et al., 2017 ) Tardive dyskinesia occurs in up to 32% of people with schizophrenia exposed to dopamine receptor antagonists.(Solmi et al., 2025 ) TD has a substantial impact on people, including leading to accidental injury, increased morbidity and mortality, as well as stigma and secondary depression.(Caroff et al., 2011 ; Chong et al., 2009 ; Solmi et al., 2025 ) Up to 50% of cases of TD are irreversible despite discontinuation of the causative drug,(Salem et al., 2017 ) highlighting the need to understand its pathophysiology, to inform the development of new treatments. One of the consequences of chronic D2 blockade is thought to be excess dopaminergic activity in the basal ganglia(Marchand & Dilda, 2006 ; Salem et al., 2017 ). Most antipsychotics have much lower affinity for dopamine D1 receptors than for D2 receptors.(Kaar et al., 2020 ) Thus, excess dopaminergic activity in the basal ganglia is thought to lead to excess activity at dopamine D1 receptors to lead to the dyskinetic movements seen in TD.(Ellison et al., 1988 ) However, to our knowledge, no prior study has investigated whether there is in vivo evidence of increased dopamine production in people with TD. To address this, we conducted an imaging study using neuromelanin-sensitive magnetic resonance imaging (NM-MRI). This provides an in vivo marker of long-term dopamine activity by measuring neuromelanin, a byproduct of dopamine turnover, in the substantia nigra-ventral tegmental area (SN-VTA).(Cassidy et al., 2019 ; Vano et al., 2024 ) Based on the hypothesis that TD is the result of long-term dopamine overactivity, we predicted that SN-VTA neuromelanin levels would be higher in patients with schizophrenia and TD relative to people with schizophrenia without TD. Methods Participants The study includes data from participants recruited across several ongoing studies approved by National Health Service (NHS) research ethics committees. All patients were recruited from community mental health teams in London, UK. Written, informed consent was obtained from all participants prior to participation. The inclusion criterion for the TD group was a DSM-V diagnosis of TD(American Psychiatric Association, 2013 ), based on clinical history and physical examination. Non-TD group participants were required to have no history of TD and no evidence of TD on examination. Additional inclusion criteria for both groups were age 18–65, and meeting criteria for a DSM-5 schizophrenia spectrum diagnosis (identified via a structured clinical interview and/or review of clinical notes). All psychotropic medication had to be prescribed at a stable dose for at least 4 weeks prior to participation. Exclusion criteria for both groups included non-tardive neurological disorders, acute psychiatric crisis, substance dependence and contraindications to MRI scanning. Participants enrolled in the study underwent a baseline assessment to confirm clinical stability and medication adherence. We collected information on demographic factors, duration of psychotic illness and antipsychotic medication (converted to olanzapine equivalents using the defined daily doses [DDD] method)(Leucht et al., 2016 ). Participants were assessed for TD and other neurological symptoms. TD symptom severity was quantified with a shortened Abnormal Involuntary Movement Scale (AIMS), rating dyskinesia on a 0–4 scale for four facial and oral areas, upper limbs, lower limbs and trunk movements. MRI acquisition Participants underwent MRI on the same 3T scanner (MAGNETOM Prisma, Siemens Healthineers) using a 64-channel receive head coil. Structural 3-dimensional T1-weighted Magnetisation Prepared - RApid Gradient Echo (MP-RAGE) images were collected (acquisition time = 05:35 − 07:16min, repetition time (TR) = 2300-2400ms, echo time (TE) = 2.22-2.91ms, flip angle = 8-9 o , bandwidth = 140-220Hz/pixel, acceleration factor 2, slices 176–192). Axial 2-dimensional gradient echo images with a magnetization transfer pulse for the NM-MRI built from a standard Siemens sequence were acquired as well (acquisition time = 5:50 − 07:18min, echo time (TE) = 2.85–3.47, flip angle = 40 o , bandwidth = 320Hz/pixel, slices = 12, voxel size = 0.75x0.75x2.00mm 3 ). Starting TR for the NM sequence was 359ms. When the specific absorption rate (SAR) was calculated to exceed scanner limits for a participant, the TR was increased to lower the SAR (up to a maximum of 702ms). NM-MRI volume placement and quality control were carried out as outlined by Salzman et al.(Salzman et al., 2021 ) Neuromelanin analysis T1 and NM-MRI processing was completed via an automated pipeline (available at https://github.com/lukevano/KCL_Neuromelanin-MRI ) based on the methods originally described by Cassidy et al ( 2019 ).(Cassidy et al., 2019 ) The full methods for the current analysis are outlined by Vano et al.(Vano et al., 2024 ). Briefly, each participant’s NM-MRI image was co-registered to their T1-weighted image and then their T1-weigted image was normalised to the Montreal Neurological Institute (MNI)(Mazziotta et al., 1995 ) space, both done via Advanced Normalization Tools 2.4.0 (ANTs)(Avants et al., 2011 ). Output from these steps was used to normalize each participant’s NM-MRI to the MNI space as well. A midbrain atlas containing the SN-VTA and crus cerebri (available at https://github.com/lukevano/KCL_Neuromelanin-MRI ) was then moved from MNI to participant’s space for each MRI image. Two independent investigators (BRB and LJV) carried out quality control for original images, normalised NM-MRI images, as well as the midbrain atlas transformation, according to criteria by Salzman et al(Salzman et al., 2021 ). Discrepancies were resolved by a third independent investigator (JS). Each masked NM-MRI image was converted to a NM contrast-to noise ratio (NM-CNR) map, where voxel values were relative to the average of a control region, the crus cerebri. These maps were spatially smoothed with a 1-mm full-width at half maximum Gaussian kernel. Mean NM-CNR for the SN-VTA was calculated for each participant by averaging voxel values within the SN-VTA mask. Statistical Analysis Analysis was conducted using in-house Python scripts. Group differences in continuous variables were assessed with Welch’s independent sample t-tests and Hegde’s g, whereas Chi-squared tests were used for categorical variables. Correlations were quantified with Pearson’s r. Cook’s distance was used to determine the influence of potential outliers on these correlations. We used an analysis of covariance (ANCOVA), implemented as an ordinary least squares regression, to assess if case-control differences were significant when controlling for variables that might influence neuromelanin measures (age, sex, psychotic illness duration, olanzapine-equivalent antipsychotic dose, SN-VTA volume). (Vano et al., 2024 ; Wengler et al., 2024 ) As acquisition TR values of the NM MRI sequence were variable, we carried out an exploratory sensitivity analysis including TR as a covariate. Model significance was assessed using Type II sums of squares. A voxelwise analysis was carried out via the spatial extent method, consistent with prior NM-CNR analyses.(Cassidy et al., 2019 ; Vano et al., 2024 ) Differences between the TD and non-TD groups were identified at a voxel-level one-sided threshold of p < 0.05. Hypothesis testing involved comparing the number of identified voxels of significant difference to that generated from a null distribution of random permutations of the case-control status (p < 0.05 with 10,000 permutations).(Cassidy et al., 2019 ; Vano et al., 2024 ) For an exploratory analysis, we used a more stringent voxel-level threshold of p < 0.01. Results Participant Details Out of the combined study cohorts with required neuroimaging measures (n = 109), thirteen participants met the inclusion criteria for the TD group. As the mean age of the TD group was greater than the mean across the cohorts, the closest age-matched non-TD controls were selected (n = 14). Therefore, data from a total of twenty-seven participants with schizophrenia spectrum disorder were used in the analysis, 13 in the TD and 14 in the non-TD group. The two groups did not significantly differ in age, sex, ethnicity, illness duration and current antipsychotic dose (see Table 1 ). Table 1 Baseline characteristics as compared between the two participant groups. Characteristic Participants with TD (n = 13) Participants without TD (n = 14) Chi 2 or t-statistic p-value N % N % Male 11 84.62% 12 85.71% 0.01 0.94 Ethnicity White 4 30.77% 5 35.71% 0.07 0.79 Non-White 9 69.23% 9 64.29% Mean SD Mean SD Age 47.31 12.63 45.86 9.21 0.74 0.34 Olanzapine dose equivalents (mg/day) 14.54 7.03 13.28 5.68 0.62 0.51 Duration of Psychotic Illness (years) 17.31 9.79 15.07 12.52 0.52 0.61 AIMS Total Score 7.69 4.19 NA NA NA NA NM-CNR NM-CNR was significantly higher in the tardive dyskinesia group (mean = 0.253), as compared to those without TD (mean = 0.223; t(17.27) = 3.817, p = 0.001; g = 1.386, 95% CI=[0.559, 2.213]); Fig. 1). Our sensitivity analysis showed that this group difference remained significant when controlling for age, sex, SN-VTA volume, olanzapine dose equivalence, and psychotic illness duration (ANCOVA: F(1,20) = 14.13, p = 0.0012; adjusted β = 0.0308, 95% CI [0.0137, 0.0478]), and also when including acquisition TR as a covariate (ANCOVA: F(1,19) = 11.21, p = 0.0034; adjusted β = 0.0299, 95% CI [0.0112, 0.0485]). SN-VTA NM-CNR itself was not significantly correlated with age (r = 0.025, p = 0.90), sex (t= -0.5491, p-value = 0.59), olanzapine dose equivalence (r = 0.208, p = 0.30), or psychotic illness duration (r = 0.160, p = 0.42). Figure 1. (A) Bar graph showing mean Neuromelanin Contrast:Noise Ratio (NM-CNR) in the Substantia Nigra – Ventral Tegmental Area (SN-VT)A compared between groups with (SCZ + TD) and without Tardive Dyskinesia (SCZ-TD); error bars = SEM. (B) Voxels where TD status was associated with significantly higher NM-CNR at a one-sided voxel-level threshold of p < 0.01 than the non-TD group are coloured in green (permutation p < 0.001). The voxel-wise analysis identified 814 of 1790 voxels where NM-CNR was significantly higher in the TD vs. the non-TD group at the voxel-level threshold p < 0.05 and 463 voxels at the voxel-level threshold p < 0.01. In both cases, the observed spatial extent exceeded that expected under the null distribution (permutation p < 0.001). The peak t score was located in the right ventral substantia nigra (t = 5.33, MNI coordinates x = 9, y=-17, z=-15). In contrast, only three voxels showed higher NM-CNR in the non-TD than the TD group at p < 0.05 but the spatial extent was not significant on permutation testing (p = 1). In the full TD sample (n = 13), NM-CNR was not significantly correlated with the total score on the AIMS (Pearson r = 0.129, p = 0.676). However, visual inspection of the data identified two clear bivariate outliers. One of these significantly overinfluenced the correlation, as indicated by Cook’s distance (Cook’s distance = 0.618, threshold = 0.308). Their outlier status may be explained by the fact that even though they met diagnostic criteria for tardive dyskinesia, a significant clinical component of their tardive syndrome was dystonia, which is not well captured by the AIMS. The second outlier was subsequently found to be a frequent user of cannabis, despite not meeting the formal criteria for substance dependence, which may be associated with higher NM-CNR values.(Ahrens et al., 2025 ) After excluding these two outliers, this exploratory correlation was highly significant (Pearson r = 0.903, p = 0.0001), see Fig. 2 . Discussion Patients with schizophrenia spectrum disorders with tardive dyskinesia had significantly higher SN-VTA NM-CNR, as compared to those without TD. The difference was most pronounced in the ventral substantia nigra. While the correlation between TD severity and SN-VTA NM-CNR was not significant in the whole sample, there was a significant association between higher NM-CNR and more severe symptoms of TD following exclusion of outliers with clinically significant confounders. To our knowledge, this demonstrates for the first time that neuromelanin levels are higher in patients with TD, compared to individuals with schizophrenia spectrum disorders who had also received long-term antipsychotic treatment but had not developed TD. A strength of our study is the inclusion of a comparison group of patients without TD but who had schizophrenia and had received comparable long-term antipsychotic treatment, controlling for the effects of illness and medication. However, it remains to be determined if TD seen in people taking antipsychotics for other conditions is also associated with higher neuromelanin levels. While our study demonstrated that the relationship between NM-CNR and TD diagnosis is significant even when age, sex and psychotic illness duration are accounted for, there are additional factors which could influence this relationship, such as schizophrenia symptom severity and socioeconomic status.(Wengler et al., 2024 ) NM-CNR was not correlated with current antipsychotic dose, but data on total lifetime antipsychotic exposure were not available. Equally, data on prior duration of TD symptoms was not available and is generally challenging to collect due to frequently delayed diagnosis. One limitation for interpretation is that we did not include healthy controls for normative comparison. However, our recent meta-analysis ( in press ) indicates that SN-VTA NM-CNR is higher in people with schizophrenia not selected for TD relative to healthy controls,(Vano et al., 2026 ) suggesting that neuromelanin may be particularly high in people with schizophrenia and TD. Interestingly, we found preliminary evidence that the peak difference in NM levels linked to TD is found within the ventral substantia nigra. NM-CNR in ventral substantia nigra has been associated with psychotic symptom severity(Cassidy et al., 2019 ; Wengler et al., 2024 ), positive response to first-line antipsychotics in schizophrenia(Van Der Pluijm et al., 2024 ), and both dopamine synthesis(Vano et al., 2024 ) and release(Cassidy et al., 2019 ) in the associative striatum. Speculatively, this could suggest that overactivity of dopamine pathways originating in the ventral substantia nigra could drive both schizophrenia and TD. However, given the exploratory nature of the voxel-wise analysis, these findings warrant further confirmation in additional studies. Interpretation of NM-CNR Neuromelanin is paramagnetic because it contains iron, meaning it can be reliably detected using MR imaging.(Cassidy et al., 2019 ) NM-CNR is directly correlated with SN-VTA neuromelanin concentrations measured post-mortem, providing concurrent validation that NM-CNR reflects neuromelanin levels(Cassidy et al., 2019 ). This is also consistent with the finding that higher NM-CNR directly correlates with higher striatal dopamine release(Cassidy et al., 2019 ) and dopamine synthesis capacity(Vano et al., 2024 ), Once produced, cytosolic dopamine in the SN-VTA has multiple cellular fates, including metabolism to neuromelanin, but also rapid sequestration by VMAT2 into vesicles (for potential release into the synapse). (Muñoz et al., 2012 ) Accordingly, when VMAT2 is overexpressed, NM accumulation is reduced.(Sulzer et al., 2000 ) Therefore, higher neuromelanin levels can result from increased production or reduced sequestration, but as VMAT2 levels in schizophrenia are unchanged(Taylor, 2000 ) or even higher(Zubieta et al., 2001 ), increased dopamine production is the most likely cause of the higher NM levels we found associated with TD. Thus, our findings most likely indicate that TD is associated with higher SN-VTA neuromelanin levels. These results are consistent with long term overactivity of mesostriatal dopamine pathways in TD. Moreover, this provides a neurobiological rationale for the use of VMAT2 inhibitors, which act to reduce dopamine levels.(Rosenthal et al., 2025 ) Implications of our findings for understanding the neurobiology of TD Because the antipsychotic drugs that cause TD can induce a homeostatic increase in subcortical dopamine signalling(Marchand & Dilda, 2006 ; Salem et al., 2017 ; Silvestri et al., 2000 ), and because effective TD treatments block dopamine packaging and subsequent dopamine release into synapses,(Huang et al., 2020 ) it has been thought that dopamine overactivity is the key pathophysiological mechanism underlying TD (Marchand & Dilda, 2006 ; Salem et al., 2017 ). This could take form of increased synthesis and release of dopamine or homeostatic D2 receptor upregulation.(Marchand & Dilda, 2006 ; Salem et al., 2017 ; Silvestri et al., 2000 ) A human PET study of D2 receptor upregulation found evidence of higher D2 density in treated vs. antipsychotic-naïve patients.(Silvestri et al., 2000 ) However, in other studies, D2R density in patients taking antipsychotics was not different between those who did and did not experience tardive dyskinesia.(Andersson et al., 1990 ; Blin et al., 1989 ) In addition, while D2R upregulation resolves within 6–8 weeks after ceasing the use antipsychotic medications,(De Beer et al., 2025 ) a large portion of TD cases appear to be irreversible (Salem et al., 2017 ). Therefore, increased dopamine synthesis and release may provide a better aetiological explanation for the emergence of TD and our results strongly support this mechanism. In vitro, antipsychotics have been shown to increase dopaminergic neuron activity in the VTA, due to feedback from postsynaptic blockade.(Valenti & Grace, 2010 ) In microdialysis studies, antipsychotic drugs have been shown to acutely facilitate dopamine release, and those with a lower propensity for extrapyramidal side effects caused a more spatially restricted pattern of dopaminergic activation.(Tanda et al., 2015 ) Longitudinal PET studies using radiolabeled l- dopa, the precursor to dopamine, have been inconsistent; with one showing that a minimum of 5 weeks of treatment with antipsychotic treatment did not lead to an increase in striatal dopamine synthesis capacity.(Jauhar et al., 2019 ) The other found that a similar duration of treatment with haloperidol was associated with synthesis reductions.(Gründer et al., 2003 ) Inconsistencies in longitudinal PET studies of dopamine synthesis capacity in schizophrenia could suggest there are individual differences in sensitivities to the effects of antipsychotics on the dopamine system, which could explain why some people develop TD and others don’t. One question that naturally arises is whether higher midbrain neuromelanin levels result from, or precede, antipsychotic treatment in those individuals who go on to develop TD. As such, higher dopamine production could either constitute an adverse effect of treatment or a baseline vulnerability to developing TD. Our recent meta-analysis demonstrated an association between antipsychotic dose and higher NM-CNR values in patients relative to controls, supporting a causative link in which more antipsychotic use leads to increased NM-CNR.(Vano et al., 2026 ) If elevated NM-CNR predates treatment, there could be a possibility of using NM-CNR as a marker of TD risk to inform treatment decisions prior to commencing antipsychotic drugs. Longitudinal NM imaging studies of TD are needed to disambiguate these possibilities. It is important to note that alternative mechanisms have been proposed(Salem et al., 2017 ; Teo et al., 2012 ) to accommodate inconsistencies in the dopamine overactivity model serving as the comprehensive mechanism leading to TD. Pre-clinical evidence suggests relevance of both D2 and cholinergic signalling in models of TD.(Bordia et al., 2016 ) In human genetic studies, incidence of TD has been linked to polymorphisms of genes related to dopamine signalling - D2(Liou et al., 2006 ) and D3(Al Hadithy et al., 2009 ) receptors, dopamine transporter (DAT)(Zivković et al., 2013 ), but also the HTR2C serotonin receptor(Al Hadithy et al., 2009 ) and manganese superoxide dismutase, involved in mitochondrial function(Liu et al., 2010 ). Multiple magnetic resonance imaging (MRI) studies of TD were also completed, identifying white matter changes(Bai et al., 2009 ), areas of reduced grey matter(Li et al., 2013 ) and motor circuit functional dysconnectivity(Yu et al., 2021 ). It is possible that hyperactivity within the dopaminergic system is necessary, but not sufficient, for causing TD, with additional complex and poorly understood cellular processes being required to lead to TD. Conclusion Neuromelanin levels in the substantia nigra and ventral tegmental area are higher in people with tardive dyskinesia than those without TD, consistent with the hypothesis that increased dopamine signalling in the basal ganglia underlies TD. The difference was most marked in the ventral substantia nigra, suggesting the nigrostriatal pathway could be particularly vulnerable to the effects of dopamine-receptor blocking antipsychotics and a target for treatment. Future longitudinal studies are needed to determine if neuromelanin differences represent a vulnerability factor for TD or are secondary to antipsychotic treatment. Declarations Acknowledgements RAMs work is funded by a Wellcome Trust Clinical Research Career Development Fellowship (224625/Z/21/Z)and is supported by the NIHR Oxford Health Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care Disclosures of interest: The study was partially funded by Neurocrine Biosciences. BR Bukala has served as a consultant for Candesic and Relation Therapeutics. RAM has received speaker/consultancy fees from Angelini Pharma, Boehringer Ingelheim, Bristol Myers Squibb, Janssen, Karuna, Lundbeck, Newron, Otsuka, and Viatris, and co-directs a company that designs digital resources to support treatment of mental ill health. RTL, DA, and DH are full-time employees of Neurocrine Biosciences, Inc. and hold equity in the company. DH received royalties from publishing with Oxford University Press. OD Howes has received investigator-initiated research funding from and/or participated in advisory or speaker meetings organized by AbbVie, Alkermes, Angelini, Autifony, Biogen, Boehringer Ingelheim, Bristol Meyers Squibb (Karuna), Clinical Ink, Delix, Eli Lilly, Elysium, Heptares, Global Medical Education, Invicro, Janssen, Karuna, Lundbeck, Merck, Neumora, Neurocrine, Ono, Ontrack/Pangea, Otsuka, Sunovion, Teva, Recordati, Roche, Rovi, and Viatris/Mylan; he was previously a part-time employee of Lundbeck. Compliance with ethical standards: All procedures involving human participants were carried out in accordance with the Declaration of Helsinki. The studies were approved by the UK National Health Service (NHS) research ethics committees (refs. 21/LO/0312, 21/LO/0188, 20-ES-0107). Informed written consent was provided by all individual participants in the studies. 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Expert Rev Neurother 17(9):883–894. https://doi.org/10.1080/14737175.2017.1361322 Salzman G, Kim J, Horga G, Wengler K (2021) Standardized Data Acquisition for Neuromelanin-Sensitive Magnetic Resonance Imaging of the Substantia Nigra. J Visualized Experiments 175:62493. https://doi.org/10.3791/62493 Silvestri S, Seeman MV, Negrete J-C, Houle S, Shammi CM, Remington GJ, Kapur S, Zipursky RB, Wilson AA, Christensen BK, Seeman P (2000) Increased dopamine D 2 receptor binding after long-term treatment with antipsychotics in humans: A clinical PET study. Psychopharmacology 152(2):174–180. https://doi.org/10.1007/s002130000532 Solmi M, Fornaro M, Caiolo S, Lussignoli M, Caiazza C, De Prisco M, Solini N, De Bartolomeis A, Iasevoli F, Pigato G, Del Giovane C, Cipriani A, Correll CU (2025) Efficacy and acceptability of pharmacological interventions for tardive dyskinesia in people with schizophrenia or mood disorders: A systematic review and network meta-analysis. Mol Psychiatry 30(3):1207–1222. https://doi.org/10.1038/s41380-024-02733-z Sulzer D, Bogulavsky J, Larsen KE, Behr G, Karatekin E, Kleinman MH, Turro N, Krantz D, Edwards RH, Greene LA, Zecca L (2000) Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles. Proceedings of the National Academy of Sciences , 97 (22), 11869–11874. https://doi.org/10.1073/pnas.97.22.11869 Tanda G, Valentini V, De Luca MA, Perra V, Serra GP, Di Chiara G (2015) A systematic microdialysis study of dopamine transmission in the accumbens shell/core and prefrontal cortex after acute antipsychotics. Psychopharmacology 232(8):1427–1440. https://doi.org/10.1007/s00213-014-3780-2 Taylor S (2000) In Vivo Measurement of the Vesicular Monoamine Transporter in Schizophrenia. Neuropsychopharmacology 23(6):667–675. https://doi.org/10.1016/S0893-133X(00)00165-2 Teo JT, Edwards MJ, Bhatia K (2012) Tardive dyskinesia is caused by maladaptive synaptic plasticity: A hypothesis. Mov Disord 27(10):1205–1215. https://doi.org/10.1002/mds.25107 Valenti O, Grace AA (2010) Antipsychotic drug-induced increases in ventral tegmental area dopamine neuron population activity via activation of the nucleus accumbens–ventral pallidum pathway. Int J Neuropsychopharmacol 13(07):845–860. https://doi.org/10.1017/S1461145709990599 Van Der Pluijm M, Wengler K, Reijers PN, Cassidy CM, Tjin Joe T, De Peuter K, Horga OR, Booij G, De Haan J, L., Van De Giessen E (2024) Neuromelanin-Sensitive MRI as Candidate Marker for Treatment Resistance in First-Episode Schizophrenia. 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Biol Psychiatry 96(8):674–683. https://doi.org/10.1016/j.biopsych.2024.06.013 Vano LJ, Sedlacik J, Carr RW, Bukala BR, Howes OD, McCutcheon RA (2026) Grey Matter Iron and Neuromelanin in Psychosis: A Systematic Review and Meta-Analysis of MRI Studies . https://doi.org/10.64898/2026.01.15.26344182 Wengler K, Baker SC, Velikovskaya A, Fogelson A, Girgis RR, Reyes-Madrigal F, Lee S, De La Fuente-Sandoval C, Ojeil N, Horga G (2024) Generalizability and Out-of-Sample Predictive Ability of Associations Between Neuromelanin-Sensitive Magnetic Resonance Imaging and Psychosis in Antipsychotic-Free Individuals. JAMA Psychiatry 81(2):198. https://doi.org/10.1001/jamapsychiatry.2023.4305 Yu T, Li Y, Li N, Huang J, Fan F, Luo X, Tan S, Yang F, Tian B, Tian L, Hong E, L., Tan Y (2021) Abnormal functional connectivity of motor circuit in the schizophrenic patients with tardive dyskinesia: A resting-state fMRI study. Neurosci Lett 742:135548. https://doi.org/10.1016/j.neulet.2020.135548 Zivković M, Mihaljević-Peles A, Bozina N, Sagud M, Nikolac-Perkovic M, Vuksan-Cusa B, Muck-Seler D (2013) The Association Study of Polymorphisms in DAT, DRD2, and COMT Genes and Acute Extrapyramidal Adverse Effects in Male Schizophrenic Patients Treated With Haloperidol. J Clin Psychopharmacol 33(5):593–599. https://doi.org/10.1097/JCP.0b013e31829abec9 Zubieta J-K, Taylor SF, Huguelet P, Koeppe RA, Kilbourn MR, Frey KA (2001) Vesicular monoamine transporter concentrations in bipolar disorder type I, schizophrenia, and healthy subjects. Biol Psychiatry 49(2):110–116. https://doi.org/10.1016/S0006-3223(00)00981-1 Additional Declarations Competing interest reported. The study was partially funded by Neurocrine Biosciences. BR Bukala has served as a consultant for Candesic and Relation Therapeutics. RAM has received speaker/consultancy fees from Angelini Pharma, Boehringer Ingelheim, Bristol Myers Squibb, Janssen, Karuna, Lundbeck, Newron, Otsuka, and Viatris, and co-directs a company that designs digital resources to support treatment of mental ill health. RTL, DA, and DH are full-time employees of Neurocrine Biosciences, Inc. and hold equity in the company. DH received royalties from publishing with Oxford University Press. OD Howes has received investigator-initiated research funding from and/or participated in advisory or speaker meetings organized by AbbVie, Alkermes, Angelini, Autifony, Biogen, Boehringer Ingelheim, Bristol Meyers Squibb (Karuna), Clinical Ink, Delix, Eli Lilly, Elysium, Heptares, Global Medical Education, Invicro, Janssen, Karuna, Lundbeck, Merck, Neumora, Neurocrine, Ono, Ontrack/Pangea, Otsuka, Sunovion, Teva, Recordati, Roche, Rovi, and Viatris/Mylan; he was previously a part-time employee of Lundbeck. 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Bukala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIie2RsUrEQBCGJwQ2TcB2Qw6fYUMg65Jwz7JB2LyChUUgcDartvcwVwQGYqdt4AqvSmWRSiKKuEcsvGLvtLPYr5kfho/5lwVwOP4hbB6+CQRgBwWD4GBzSpGgmMl/U/C0woPbYfe2yYFTVY7y+ik9a8AbJ8DUpgj9wBM9VCDWCqnsthlF8CMNmFmL9YrQsEUTqppKsi2YKRYDYGFVngcSfcxKM8nPx73ivx9VekLi+YrqaLlqM6OQ/RVrMaEViRdtFQo9qIvy7jKl6K2EZpX1+TzoSPTS5uc8UGk/vi6T+5sG++kqT2qb8034I3v1kV9xOBwOx2/4AlwNTt657fl1AAAAAElFTkSuQmCC","orcid":"","institution":"King’s College London","correspondingAuthor":true,"prefix":"","firstName":"Bernard","middleName":"R.","lastName":"Bukala","suffix":""},{"id":629885127,"identity":"d9e3e849-ac90-4d20-89f3-cd5656313981","order_by":1,"name":"Luke J. Vano","email":"","orcid":"","institution":"King’s College London","correspondingAuthor":false,"prefix":"","firstName":"Luke","middleName":"J.","lastName":"Vano","suffix":""},{"id":629885129,"identity":"cc84903a-f943-4199-9be7-3e41fd5e64a0","order_by":2,"name":"Richard Carr","email":"","orcid":"","institution":"King’s College London","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Carr","suffix":""},{"id":629885130,"identity":"3d3e2cc0-3b04-43a3-b951-b2cd7ed889c8","order_by":3,"name":"Alistair Cannon","email":"","orcid":"","institution":"King’s College London","correspondingAuthor":false,"prefix":"","firstName":"Alistair","middleName":"","lastName":"Cannon","suffix":""},{"id":629885131,"identity":"fd309fc9-07ab-43cf-b0f9-bb0bd5517363","order_by":4,"name":"Connor Cummings","email":"","orcid":"","institution":"King’s College London","correspondingAuthor":false,"prefix":"","firstName":"Connor","middleName":"","lastName":"Cummings","suffix":""},{"id":629885132,"identity":"3f96ab2a-0404-4dcb-bcb8-4a4c417a5c94","order_by":5,"name":"David Davies","email":"","orcid":"","institution":"Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Davies","suffix":""},{"id":629885133,"identity":"88cf104e-ddfa-463e-9c63-1fd50182791a","order_by":6,"name":"Robert McCutcheon","email":"","orcid":"","institution":"King’s College London","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"McCutcheon","suffix":""},{"id":629885134,"identity":"56581c74-9b5d-4cfb-ad4a-23cb10c71ce6","order_by":7,"name":"Ben Statton","email":"","orcid":"","institution":"Mansfield Centre for Innovation - MR Facility, Hammersmith Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Statton","suffix":""},{"id":629885135,"identity":"21e7357d-91d6-47de-b00a-16306f06e771","order_by":8,"name":"Alaine Berry","email":"","orcid":"","institution":"Mansfield Centre for Innovation - MR Facility, Hammersmith Hospital","correspondingAuthor":false,"prefix":"","firstName":"Alaine","middleName":"","lastName":"Berry","suffix":""},{"id":629885136,"identity":"ac8a59e3-7927-4212-a57c-fc8b9965dd7a","order_by":9,"name":"Jan Sedlacik","email":"","orcid":"","institution":"Mansfield Centre for Innovation - MR Facility, Hammersmith Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Sedlacik","suffix":""},{"id":629885137,"identity":"e79c1b1c-e17c-4396-8d77-f687c4389797","order_by":10,"name":"Daniel Albrecht","email":"","orcid":"","institution":"Neurocrine Biosciences, Inc","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Albrecht","suffix":""},{"id":629885138,"identity":"fcb76c6d-3c5e-4399-a9d5-8b6f9f3e5cbb","order_by":11,"name":"Ryan Terry-Lorenzo","email":"","orcid":"","institution":"Neurocrine Biosciences, Inc","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Terry-Lorenzo","suffix":""},{"id":629885139,"identity":"293bb15a-0fbc-4a0d-8eb8-bb339e784f1f","order_by":12,"name":"Dietrich Haubenberger","email":"","orcid":"","institution":"Neurocrine Biosciences, Inc","correspondingAuthor":false,"prefix":"","firstName":"Dietrich","middleName":"","lastName":"Haubenberger","suffix":""},{"id":629885140,"identity":"5399a872-eeaf-4226-8105-d20d9872feb6","order_by":13,"name":"Oliver D. Howes","email":"","orcid":"","institution":"King’s College London","correspondingAuthor":false,"prefix":"","firstName":"Oliver","middleName":"D.","lastName":"Howes","suffix":""}],"badges":[],"createdAt":"2026-03-27 13:42:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9245393/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9245393/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108391040,"identity":"abed182e-fa7c-453b-91a6-8c572f751a33","added_by":"auto","created_at":"2026-05-04 07:03:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":846345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e(A) Bar graph showing mean Neuromelanin Contrast:Noise Ratio (NM-CNR) in the Substantia Nigra – Ventral Tegmental Area (SN-VT)A compared between groups with (SCZ+TD) and without Tardive Dyskinesia (SCZ-TD); error bars = SEM. (B) Voxels where TD status was associated with significantly higher NM-CNR at a one-sided voxel-level threshold of p\u0026lt;0.01 than the non-TD group are coloured in green (permutation p\u0026lt;0.001).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9245393/v1/3dddd5d8b2f689967120ef3b.png"},{"id":108492564,"identity":"ee12e324-bbd2-41d2-8793-04060460965b","added_by":"auto","created_at":"2026-05-05 09:58:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":359189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCorrelation of tardive dyskinesia symptoms, as measured by the AIMS, with SN-VTA NM-CNR in the full sample (Left) and after exclusion of two bivariate outliers (Right).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9245393/v1/ea00bad2f5c1490b81f1e05f.png"},{"id":108803711,"identity":"5cc6480d-a315-4958-8734-465017d26bd5","added_by":"auto","created_at":"2026-05-08 15:04:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1987217,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9245393/v1/8cb07d8c-5ac3-45e8-b3ad-9281bc87b46b.pdf"}],"financialInterests":"Competing interest reported. The study was partially funded by Neurocrine Biosciences. \nBR Bukala has served as a consultant for Candesic and Relation Therapeutics. \nRAM has received speaker/consultancy fees from Angelini Pharma, Boehringer Ingelheim, Bristol Myers Squibb, Janssen, Karuna, Lundbeck, Newron, Otsuka, and Viatris, and co-directs a company that designs digital resources to support treatment of mental ill health.\nRTL, DA, and DH are full-time employees of Neurocrine Biosciences, Inc. and hold equity in the company. DH received royalties from publishing with Oxford University Press.\nOD Howes has received investigator-initiated research funding from and/or participated in advisory or speaker meetings organized by AbbVie, Alkermes, Angelini, Autifony, Biogen, Boehringer Ingelheim, Bristol Meyers Squibb (Karuna), Clinical Ink, Delix, Eli Lilly, Elysium, Heptares, Global Medical Education, Invicro, Janssen, Karuna, Lundbeck, Merck, Neumora, Neurocrine, Ono, Ontrack/Pangea, Otsuka, Sunovion, Teva, Recordati, Roche, Rovi, and Viatris/Mylan; he was previously a part-time employee of Lundbeck.","formattedTitle":"The mechanism underlying tardive dyskinesia: an exploratory test of the dopamine overactivity hypothesis using MRI imaging of midbrain neuromelanin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTardive dyskinesia (TD) is a neurological syndrome characterised by involuntary, repetitive, non-rhythmic movements.(Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) It commonly presents with orofacial symptoms, but can also involve limbs, digits, and truncal, pharyngeal or diaphragmatic muscles.(American Psychiatric Association, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) TD results from exposure to anti-dopaminergic drugs, most often antipsychotics, and symptoms usually emerge after at least a few months of treatment.(American Psychiatric Association, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) Tardive dyskinesia occurs in up to 32% of people with schizophrenia exposed to dopamine receptor antagonists.(Solmi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) TD has a substantial impact on people, including leading to accidental injury, increased morbidity and mortality, as well as stigma and secondary depression.(Caroff et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Chong et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Solmi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) Up to 50% of cases of TD are irreversible despite discontinuation of the causative drug,(Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) highlighting the need to understand its pathophysiology, to inform the development of new treatments.\u003c/p\u003e \u003cp\u003eOne of the consequences of chronic D2 blockade is thought to be excess dopaminergic activity in the basal ganglia(Marchand \u0026amp; Dilda, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Most antipsychotics have much lower affinity for dopamine D1 receptors than for D2 receptors.(Kaar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Thus, excess dopaminergic activity in the basal ganglia is thought to lead to excess activity at dopamine D1 receptors to lead to the dyskinetic movements seen in TD.(Ellison et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) However, to our knowledge, no prior study has investigated whether there is in vivo evidence of increased dopamine production in people with TD. To address this, we conducted an imaging study using neuromelanin-sensitive magnetic resonance imaging (NM-MRI). This provides an in vivo marker of long-term dopamine activity by measuring neuromelanin, a byproduct of dopamine turnover, in the substantia nigra-ventral tegmental area (SN-VTA).(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Based on the hypothesis that TD is the result of long-term dopamine overactivity, we predicted that SN-VTA neuromelanin levels would be higher in patients with schizophrenia and TD relative to people with schizophrenia without TD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eThe study includes data from participants recruited across several ongoing studies approved by National Health Service (NHS) research ethics committees. All patients were recruited from community mental health teams in London, UK. Written, informed consent was obtained from all participants prior to participation. The inclusion criterion for the TD group was a DSM-V diagnosis of TD(American Psychiatric Association, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), based on clinical history and physical examination. Non-TD group participants were required to have no history of TD and no evidence of TD on examination. Additional inclusion criteria for both groups were age 18\u0026ndash;65, and meeting criteria for a DSM-5 schizophrenia spectrum diagnosis (identified via a structured clinical interview and/or review of clinical notes). All psychotropic medication had to be prescribed at a stable dose for at least 4 weeks prior to participation. Exclusion criteria for both groups included non-tardive neurological disorders, acute psychiatric crisis, substance dependence and contraindications to MRI scanning. Participants enrolled in the study underwent a baseline assessment to confirm clinical stability and medication adherence. We collected information on demographic factors, duration of psychotic illness and antipsychotic medication (converted to olanzapine equivalents using the defined daily doses [DDD] method)(Leucht et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Participants were assessed for TD and other neurological symptoms. TD symptom severity was quantified with a shortened Abnormal Involuntary Movement Scale (AIMS), rating dyskinesia on a 0\u0026ndash;4 scale for four facial and oral areas, upper limbs, lower limbs and trunk movements.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMRI acquisition\u003c/h3\u003e\n\u003cp\u003eParticipants underwent MRI on the same 3T scanner (MAGNETOM Prisma, Siemens Healthineers) using a 64-channel receive head coil. Structural 3-dimensional T1-weighted Magnetisation Prepared - RApid Gradient Echo (MP-RAGE) images were collected (acquisition time\u0026thinsp;=\u0026thinsp;05:35\u0026thinsp;\u0026minus;\u0026thinsp;07:16min, repetition time (TR)\u0026thinsp;=\u0026thinsp;2300-2400ms, echo time (TE)\u0026thinsp;=\u0026thinsp;2.22-2.91ms, flip angle\u0026thinsp;=\u0026thinsp;8-9\u003csup\u003eo\u003c/sup\u003e, bandwidth\u0026thinsp;=\u0026thinsp;140-220Hz/pixel, acceleration factor 2, slices 176\u0026ndash;192). Axial 2-dimensional gradient echo images with a magnetization transfer pulse for the NM-MRI built from a standard Siemens sequence were acquired as well (acquisition time\u0026thinsp;=\u0026thinsp;5:50\u0026thinsp;\u0026minus;\u0026thinsp;07:18min, echo time (TE)\u0026thinsp;=\u0026thinsp;2.85\u0026ndash;3.47, flip angle\u0026thinsp;=\u0026thinsp;40\u003csup\u003eo\u003c/sup\u003e, bandwidth\u0026thinsp;=\u0026thinsp;320Hz/pixel, slices\u0026thinsp;=\u0026thinsp;12, voxel size\u0026thinsp;=\u0026thinsp;0.75x0.75x2.00mm\u003csup\u003e3\u003c/sup\u003e). Starting TR for the NM sequence was 359ms. When the specific absorption rate (SAR) was calculated to exceed scanner limits for a participant, the TR was increased to lower the SAR (up to a maximum of 702ms). NM-MRI volume placement and quality control were carried out as outlined by Salzman et al.(Salzman et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eNeuromelanin analysis\u003c/h3\u003e\n\u003cp\u003eT1 and NM-MRI processing was completed via an automated pipeline (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/lukevano/KCL_Neuromelanin-MRI\u003c/span\u003e\u003cspan address=\"https://github.com/lukevano/KCL_Neuromelanin-MRI\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on the methods originally described by Cassidy et al (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) The full methods for the current analysis are outlined by Vano et al.(Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, each participant\u0026rsquo;s NM-MRI image was co-registered to their T1-weighted image and then their T1-weigted image was normalised to the Montreal Neurological Institute (MNI)(Mazziotta et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) space, both done via Advanced Normalization Tools 2.4.0 (ANTs)(Avants et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Output from these steps was used to normalize each participant\u0026rsquo;s NM-MRI to the MNI space as well. A midbrain atlas containing the SN-VTA and crus cerebri (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/lukevano/KCL_Neuromelanin-MRI\u003c/span\u003e\u003cspan address=\"https://github.com/lukevano/KCL_Neuromelanin-MRI\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was then moved from MNI to participant\u0026rsquo;s space for each MRI image. Two independent investigators (BRB and LJV) carried out quality control for original images, normalised NM-MRI images, as well as the midbrain atlas transformation, according to criteria by Salzman et al(Salzman et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Discrepancies were resolved by a third independent investigator (JS). Each masked NM-MRI image was converted to a NM contrast-to noise ratio (NM-CNR) map, where voxel values were relative to the average of a control region, the crus cerebri. These maps were spatially smoothed with a 1-mm full-width at half maximum Gaussian kernel. Mean NM-CNR for the SN-VTA was calculated for each participant by averaging voxel values within the SN-VTA mask.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAnalysis was conducted using in-house Python scripts. Group differences in continuous variables were assessed with Welch\u0026rsquo;s independent sample t-tests and Hegde\u0026rsquo;s g, whereas Chi-squared tests were used for categorical variables. Correlations were quantified with Pearson\u0026rsquo;s r. Cook\u0026rsquo;s distance was used to determine the influence of potential outliers on these correlations.\u003c/p\u003e \u003cp\u003eWe used an analysis of covariance (ANCOVA), implemented as an ordinary least squares regression, to assess if case-control differences were significant when controlling for variables that might influence neuromelanin measures (age, sex, psychotic illness duration, olanzapine-equivalent antipsychotic dose, SN-VTA volume). (Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wengler et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) As acquisition TR values of the NM MRI sequence were variable, we carried out an exploratory sensitivity analysis including TR as a covariate. Model significance was assessed using Type II sums of squares.\u003c/p\u003e \u003cp\u003eA voxelwise analysis was carried out via the spatial extent method, consistent with prior NM-CNR analyses.(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Differences between the TD and non-TD groups were identified at a voxel-level one-sided threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Hypothesis testing involved comparing the number of identified voxels of significant difference to that generated from a null distribution of random permutations of the case-control status (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 with 10,000 permutations).(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) For an exploratory analysis, we used a more stringent voxel-level threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eParticipant Details\u003c/h2\u003e \u003cp\u003eOut of the combined study cohorts with required neuroimaging measures (n\u0026thinsp;=\u0026thinsp;109), thirteen participants met the inclusion criteria for the TD group. As the mean age of the TD group was greater than the mean across the cohorts, the closest age-matched non-TD controls were selected (n\u0026thinsp;=\u0026thinsp;14). Therefore, data from a total of twenty-seven participants with schizophrenia spectrum disorder were used in the analysis, 13 in the TD and 14 in the non-TD group. The two groups did not significantly differ in age, sex, ethnicity, illness duration and current antipsychotic dose (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics as compared between the two participant groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eParticipants with TD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eParticipants without TD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChi\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eor\u003c/p\u003e \u003cp\u003et-statistic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84.62%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.71%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthnicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.77%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.71%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-White\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.23%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.29%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlanzapine dose equivalents (mg/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of Psychotic Illness (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAIMS Total Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNM-CNR\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNM-CNR was significantly higher in the tardive dyskinesia group (mean\u0026thinsp;=\u0026thinsp;0.253), as compared to those without TD (mean\u0026thinsp;=\u0026thinsp;0.223; t(17.27)\u0026thinsp;=\u0026thinsp;3.817, p\u0026thinsp;=\u0026thinsp;0.001; g\u0026thinsp;=\u0026thinsp;1.386, 95% CI=[0.559, 2.213]); Fig.\u0026nbsp;1). Our sensitivity analysis showed that this group difference remained significant when controlling for age, sex, SN-VTA volume, olanzapine dose equivalence, and psychotic illness duration (ANCOVA: F(1,20)\u0026thinsp;=\u0026thinsp;14.13, p\u0026thinsp;=\u0026thinsp;0.0012; adjusted β\u0026thinsp;=\u0026thinsp;0.0308, 95% CI [0.0137, 0.0478]), and also when including acquisition TR as a covariate (ANCOVA: F(1,19)\u0026thinsp;=\u0026thinsp;11.21, p\u0026thinsp;=\u0026thinsp;0.0034; adjusted β\u0026thinsp;=\u0026thinsp;0.0299, 95% CI [0.0112, 0.0485]). SN-VTA NM-CNR itself was not significantly correlated with age (r\u0026thinsp;=\u0026thinsp;0.025, p\u0026thinsp;=\u0026thinsp;0.90), sex (t= -0.5491, p-value\u0026thinsp;=\u0026thinsp;0.59), olanzapine dose equivalence (r\u0026thinsp;=\u0026thinsp;0.208, p\u0026thinsp;=\u0026thinsp;0.30), or psychotic illness duration (r\u0026thinsp;=\u0026thinsp;0.160, p\u0026thinsp;=\u0026thinsp;0.42).\u003c/p\u003e \u003cp\u003e \u003cem\u003eFigure 1. (A) Bar graph showing mean Neuromelanin Contrast:Noise Ratio (NM-CNR) in the Substantia Nigra \u0026ndash; Ventral Tegmental Area (SN-VT)A compared between groups with (SCZ\u0026thinsp;+\u0026thinsp;TD) and without Tardive Dyskinesia (SCZ-TD); error bars\u0026thinsp;=\u0026thinsp;SEM. (B) Voxels where TD status was associated with significantly higher NM-CNR at a one-sided voxel-level threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 than the non-TD group are coloured in green (permutation p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe voxel-wise analysis identified 814 of 1790 voxels where NM-CNR was significantly higher in the TD vs. the non-TD group at the voxel-level threshold p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 463 voxels at the voxel-level threshold p\u0026thinsp;\u0026lt;\u0026thinsp;0.01. In both cases, the observed spatial extent exceeded that expected under the null distribution (permutation p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The peak t score was located in the right ventral substantia nigra (t\u0026thinsp;=\u0026thinsp;5.33, MNI coordinates x\u0026thinsp;=\u0026thinsp;9, y=-17, z=-15). In contrast, only three voxels showed higher NM-CNR in the non-TD than the TD group at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 but the spatial extent was not significant on permutation testing (p\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003cp\u003eIn the full TD sample (n\u0026thinsp;=\u0026thinsp;13), NM-CNR was not significantly correlated with the total score on the AIMS (Pearson r\u0026thinsp;=\u0026thinsp;0.129, p\u0026thinsp;=\u0026thinsp;0.676). However, visual inspection of the data identified two clear bivariate outliers. One of these significantly overinfluenced the correlation, as indicated by Cook\u0026rsquo;s distance (Cook\u0026rsquo;s distance\u0026thinsp;=\u0026thinsp;0.618, threshold\u0026thinsp;=\u0026thinsp;0.308). Their outlier status may be explained by the fact that even though they met diagnostic criteria for tardive dyskinesia, a significant clinical component of their tardive syndrome was dystonia, which is not well captured by the AIMS. The second outlier was subsequently found to be a frequent user of cannabis, despite not meeting the formal criteria for substance dependence, which may be associated with higher NM-CNR values.(Ahrens et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) After excluding these two outliers, this exploratory correlation was highly significant (Pearson r\u0026thinsp;=\u0026thinsp;0.903, p\u0026thinsp;=\u0026thinsp;0.0001), see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePatients with schizophrenia spectrum disorders with tardive dyskinesia had significantly higher SN-VTA NM-CNR, as compared to those without TD. The difference was most pronounced in the ventral substantia nigra. While the correlation between TD severity and SN-VTA NM-CNR was not significant in the whole sample, there was a significant association between higher NM-CNR and more severe symptoms of TD following exclusion of outliers with clinically significant confounders. To our knowledge, this demonstrates for the first time that neuromelanin levels are higher in patients with TD, compared to individuals with schizophrenia spectrum disorders who had also received long-term antipsychotic treatment but had not developed TD.\u003c/p\u003e \u003cp\u003eA strength of our study is the inclusion of a comparison group of patients without TD but who had schizophrenia and had received comparable long-term antipsychotic treatment, controlling for the effects of illness and medication. However, it remains to be determined if TD seen in people taking antipsychotics for other conditions is also associated with higher neuromelanin levels. While our study demonstrated that the relationship between NM-CNR and TD diagnosis is significant even when age, sex and psychotic illness duration are accounted for, there are additional factors which could influence this relationship, such as schizophrenia symptom severity and socioeconomic status.(Wengler et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) NM-CNR was not correlated with current antipsychotic dose, but data on total lifetime antipsychotic exposure were not available. Equally, data on prior duration of TD symptoms was not available and is generally challenging to collect due to frequently delayed diagnosis.\u003c/p\u003e \u003cp\u003eOne limitation for interpretation is that we did not include healthy controls for normative comparison. However, our recent meta-analysis (\u003cem\u003ein press\u003c/em\u003e) indicates that SN-VTA NM-CNR is higher in people with schizophrenia not selected for TD relative to healthy controls,(Vano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2026\u003c/span\u003e) suggesting that neuromelanin may be particularly high in people with schizophrenia \u003cem\u003eand\u003c/em\u003e TD. Interestingly, we found preliminary evidence that the peak difference in NM levels linked to TD is found within the ventral substantia nigra. NM-CNR in ventral substantia nigra has been associated with psychotic symptom severity(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wengler et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), positive response to first-line antipsychotics in schizophrenia(Van Der Pluijm et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and both dopamine synthesis(Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and release(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) in the associative striatum. Speculatively, this could suggest that overactivity of dopamine pathways originating in the ventral substantia nigra could drive both schizophrenia and TD. However, given the exploratory nature of the voxel-wise analysis, these findings warrant further confirmation in additional studies.\u003c/p\u003e\n\u003ch3\u003eInterpretation of NM-CNR\u003c/h3\u003e\n\u003cp\u003eNeuromelanin is paramagnetic because it contains iron, meaning it can be reliably detected using MR imaging.(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) NM-CNR is directly correlated with SN-VTA neuromelanin concentrations measured post-mortem, providing concurrent validation that NM-CNR reflects neuromelanin levels(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This is also consistent with the finding that higher NM-CNR directly correlates with higher striatal dopamine release(Cassidy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and dopamine synthesis capacity(Vano et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), Once produced, cytosolic dopamine in the SN-VTA has multiple cellular fates, including metabolism to neuromelanin, but also rapid sequestration by VMAT2 into vesicles (for potential release into the synapse). (Mu\u0026ntilde;oz et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) Accordingly, when VMAT2 is overexpressed, NM accumulation is reduced.(Sulzer et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) Therefore, higher neuromelanin levels can result from increased production or reduced sequestration, but as VMAT2 levels in schizophrenia are unchanged(Taylor, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) or even higher(Zubieta et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), increased dopamine production is the most likely cause of the higher NM levels we found associated with TD. Thus, our findings most likely indicate that TD is associated with higher SN-VTA neuromelanin levels. These results are consistent with long term overactivity of mesostriatal dopamine pathways in TD. Moreover, this provides a neurobiological rationale for the use of VMAT2 inhibitors, which act to reduce dopamine levels.(Rosenthal et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImplications of our findings for understanding the neurobiology of TD\u003c/h2\u003e \u003cp\u003eBecause the antipsychotic drugs that cause TD can induce a homeostatic increase in subcortical dopamine signalling(Marchand \u0026amp; Dilda, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Silvestri et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and because effective TD treatments block dopamine packaging and subsequent dopamine release into synapses,(Huang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) it has been thought that dopamine overactivity is the key pathophysiological mechanism underlying TD (Marchand \u0026amp; Dilda, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This could take form of increased synthesis and release of dopamine or homeostatic D2 receptor upregulation.(Marchand \u0026amp; Dilda, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Silvestri et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) A human PET study of D2 receptor upregulation found evidence of higher D2 density in treated vs. antipsychotic-na\u0026iuml;ve patients.(Silvestri et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) However, in other studies, D2R density in patients taking antipsychotics was not different between those who did and did not experience tardive dyskinesia.(Andersson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Blin et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) In addition, while D2R upregulation resolves within 6\u0026ndash;8 weeks after ceasing the use antipsychotic medications,(De Beer et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) a large portion of TD cases appear to be irreversible (Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, increased dopamine synthesis and release may provide a better aetiological explanation for the emergence of TD and our results strongly support this mechanism.\u003c/p\u003e \u003cp\u003eIn vitro, antipsychotics have been shown to increase dopaminergic neuron activity in the VTA, due to feedback from postsynaptic blockade.(Valenti \u0026amp; Grace, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) In microdialysis studies, antipsychotic drugs have been shown to acutely facilitate dopamine release, and those with a lower propensity for extrapyramidal side effects caused a more spatially restricted pattern of dopaminergic activation.(Tanda et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Longitudinal PET studies using radiolabeled \u003cem\u003el-\u003c/em\u003edopa, the precursor to dopamine, have been inconsistent; with one showing that a minimum of 5 weeks of treatment with antipsychotic treatment did not lead to an increase in striatal dopamine synthesis capacity.(Jauhar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) The other found that a similar duration of treatment with haloperidol was associated with synthesis reductions.(Gr\u0026uuml;nder et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) Inconsistencies in longitudinal PET studies of dopamine synthesis capacity in schizophrenia could suggest there are individual differences in sensitivities to the effects of antipsychotics on the dopamine system, which could explain why some people develop TD and others don\u0026rsquo;t.\u003c/p\u003e \u003cp\u003eOne question that naturally arises is whether higher midbrain neuromelanin levels result from, or precede, antipsychotic treatment in those individuals who go on to develop TD. As such, higher dopamine production could either constitute an adverse effect of treatment or a baseline vulnerability to developing TD. Our recent meta-analysis demonstrated an association between antipsychotic dose and higher NM-CNR values in patients relative to controls, supporting a causative link in which more antipsychotic use leads to increased NM-CNR.(Vano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2026\u003c/span\u003e) If elevated NM-CNR predates treatment, there could be a possibility of using NM-CNR as a marker of TD risk to inform treatment decisions prior to commencing antipsychotic drugs. Longitudinal NM imaging studies of TD are needed to disambiguate these possibilities.\u003c/p\u003e \u003cp\u003eIt is important to note that alternative mechanisms have been proposed(Salem et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Teo et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) to accommodate inconsistencies in the dopamine overactivity model serving as the comprehensive mechanism leading to TD. Pre-clinical evidence suggests relevance of both D2 and cholinergic signalling in models of TD.(Bordia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) In human genetic studies, incidence of TD has been linked to polymorphisms of genes related to dopamine signalling - D2(Liou et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and D3(Al Hadithy et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) receptors, dopamine transporter (DAT)(Zivković et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), but also the HTR2C serotonin receptor(Al Hadithy et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and manganese superoxide dismutase, involved in mitochondrial function(Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Multiple magnetic resonance imaging (MRI) studies of TD were also completed, identifying white matter changes(Bai et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), areas of reduced grey matter(Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and motor circuit functional dysconnectivity(Yu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is possible that hyperactivity within the dopaminergic system is necessary, but not sufficient, for causing TD, with additional complex and poorly understood cellular processes being required to lead to TD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNeuromelanin levels in the substantia nigra and ventral tegmental area are higher in people with tardive dyskinesia than those without TD, consistent with the hypothesis that increased dopamine signalling in the basal ganglia underlies TD. The difference was most marked in the ventral substantia nigra, suggesting the nigrostriatal pathway could be particularly vulnerable to the effects of dopamine-receptor blocking antipsychotics and a target for treatment. Future longitudinal studies are needed to determine if neuromelanin differences represent a vulnerability factor for TD or are secondary to antipsychotic treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAMs work is funded by a Wellcome Trust Clinical Research Career Development Fellowship (224625/Z/21/Z)and is supported by the NIHR Oxford Health Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was partially funded by Neurocrine Biosciences. \u003c/p\u003e\n\u003cp\u003eBR Bukala has served as a consultant for Candesic and Relation Therapeutics. \u003c/p\u003e\n\u003cp\u003eRAM has received speaker/consultancy fees from Angelini Pharma, Boehringer Ingelheim, Bristol Myers Squibb, Janssen, Karuna, Lundbeck, Newron, Otsuka, and Viatris, and co-directs a company that designs digital resources to support treatment of mental ill health.\u003c/p\u003e\n\u003cp\u003eRTL, DA, and DH are full-time employees of Neurocrine Biosciences, Inc. and hold equity in the company. DH received royalties from publishing with Oxford University Press.\u003c/p\u003e\n\u003cp\u003eOD Howes has received investigator-initiated research funding from and/or participated in advisory or speaker meetings organized by AbbVie, Alkermes, Angelini, Autifony, Biogen, Boehringer Ingelheim, Bristol Meyers Squibb (Karuna), Clinical Ink, Delix, Eli Lilly, Elysium, Heptares, Global Medical Education, Invicro, Janssen, Karuna, Lundbeck, Merck, Neumora, Neurocrine, Ono, Ontrack/Pangea, Otsuka, Sunovion, Teva, Recordati, Roche, Rovi, and Viatris/Mylan; he was previously a part-time employee of Lundbeck.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving human participants were carried out in accordance with the Declaration of Helsinki. The studies were approved by the UK National Health Service (NHS) research ethics committees (refs. 21/LO/0312, 21/LO/0188, 20-ES-0107). Informed written consent was provided by all individual participants in the studies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhrens J, Ford SD, Schaefer B, Reese D, Khan AR, Tibbo P, Rabin R, Cassidy CM, Palaniyappan L (2025) Convergence of Cannabis and Psychosis on the Dopamine System. 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Biol Psychiatry 49(2):110\u0026ndash;116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0006-3223(00)00981-1\u003c/span\u003e\u003cspan address=\"10.1016/S0006-3223(00)00981-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Psychosis, Neuroimaging, Pharmacology","lastPublishedDoi":"10.21203/rs.3.rs-9245393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9245393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eRationale\u003c/h2\u003e \u003cp\u003eTardive dyskinesia (TD) is a neurological syndrome of involuntary repetitive movements which results from treatment with antipsychotic medication. The pathoetiology of TD is not well understood but a possible mechanism involves dopaminergic overactivity in the nigrostriatal pathway. If this theory is correct, then levels of neuromelanin (a long-term marker of dopaminergic activity) should be higher in people with TD than those without TD.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThe aim of the study was to test the hypothesis that neuromelanin levels are higher in patients with TD relative to those without TD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eData from 27 participants (TD: n\u0026thinsp;=\u0026thinsp;13; non-TD n\u0026thinsp;=\u0026thinsp;14) with a diagnosis of schizophrenia, all taking antipsychotic drugs, was used. Neuromelanin was measured in the midbrain via Magnetic Resonance Imaging (MRI) and compared between the groups. Movement symptoms were measured using the Abnormal Involuntary Movement Scale (AIMS), and antipsychotic dose recorded.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMRI-measured neuromelanin levels were significantly higher in patients with TD, as compared to those without (mean\u0026thinsp;=\u0026thinsp;0.223; t(17.27)\u0026thinsp;=\u0026thinsp;3.817, p\u0026thinsp;=\u0026thinsp;0.001; g\u0026thinsp;=\u0026thinsp;1.386, 95% CI=[0.559, 2.213]). This remained significant after controlling for age, sex, substantia nigra/ventral tegmental area volume and antipsychotic dose (ANCOVA: F(1,20)\u0026thinsp;=\u0026thinsp;12.08, p\u0026thinsp;=\u0026thinsp;0.0024; adjusted β\u0026thinsp;=\u0026thinsp;0.0301, 95% CI [0.0120, 0.0481]). The most pronounced difference was seen in the ventral substantia nigra.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe finding of higher midbrain neuromelanin in patients with TD aligns with the dopamine overactivity hypothesis of TD aetiology. It also supports a neurobiological basis for treatment of TD with drugs that target presynaptic dopamine activity, such as VMAT2 inhibitors. Additionally, it identifies the ventral substantia nigra as a key locus. Future longitudinal studies are needed to delineate if dopamine overactivity develops in response to antipsychotic treatment or is a trait vulnerability marker for risk of TD.\u003c/p\u003e","manuscriptTitle":"The mechanism underlying tardive dyskinesia: an exploratory test of the dopamine overactivity hypothesis using MRI imaging of midbrain neuromelanin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 07:03:39","doi":"10.21203/rs.3.rs-9245393/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74fe913e-dba9-46ef-b0c8-7e64e7d8800d","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-12T20:55:00+00:00","index":36,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T07:03:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 07:03:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9245393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9245393","identity":"rs-9245393","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}