Network biomarkers in recovered psychosis patients who discontinued antipsychotics.

There are no studies investigating topological properties of resting-state fMRI (rs-fMRI) in patients who have recovered from psychosis and discontinued medication (hereafter, recovered patients [RP]). This study aimed to explore topological organization of the functional brain connectome in the RP using graph theory approach. We recruited 30 RP and 50 age and sex-matched healthy controls (HC). The RP were further divided into the subjects who were relapsed after discontinuation of antipsychotics (RP-R) and who maintained recovered state without relapse (RP-M). Using graph-based network analysis of rs-fMRI signals, global and local metrics and hub information were obtained. The robustness of the network was tested with random failure and targeted attack. As an ancillary analysis, Network-Based Statistic (NBS) was performed. Association of significant findings with psychopathology and cognitive functioning was also explored. The RP showed intact network properties in terms of global and local metrics. However, higher global functional connectivity strength and hyperconnectivity in the interconnected component were observed in the RP compared to HC. In the subgroup analysis, the RP-R were found to have lower global efficiency, longer characteristic path length and lower robustness whereas no such abnormalities were identified in the RP-M. Associations of the degree centrality of some hubs with cognitive functioning were identified in the RP-M. Even though network properties of the RP were intact, subgroup analysis revealed more altered topological organizations in the RP-R. The findings in the RP-R and RP-M may serve as network biomarkers for predicting relapse or maintained recovery after the discontinuation of antipsychotics.

Diagnosis of schizophrenia with functional connectome data: a graph-based convolutional neural network approach.

Previous deep learning methods have not captured graph or network representations of brain structural or functional connectome data. To address this, we developed the BrainNet-Global Covariance Pooling-Attention Convolutional Neural Network (BrainNet-GA CNN) by incorporating BrainNetCNN and global covariance pooling into the self-attention mechanism. Resting-state functional magnetic resonance imaging data were obtained from 171 patients with schizophrenia spectrum disorders (SSDs) and 161 healthy controls (HCs). We conducted an ablation analysis of the proposed BrainNet-GA CNN and quantitative performance comparisons with competing methods using the nested tenfold cross validation strategy. The performance of our model was compared with competing methods. Discriminative connections were visualized using the gradient-based explanation method and compared with the results obtained using functional connectivity analysis. The BrainNet-GA CNN showed an accuracy of 83.13%, outperforming other competing methods. Among the top 10 discriminative connections, some were associated with the default mode network and auditory network. Interestingly, these regions were also significant in the functional connectivity analysis. Our findings suggest that the proposed BrainNet-GA CNN can classify patients with SSDs and HCs with higher accuracy than other models. Visualization of salient regions provides important clinical information. These results highlight the potential use of the BrainNet-GA CNN in the diagnosis of schizophrenia.

Longitudinal symptom network structure in first-episode psychosis: a possible marker for remission.

BACKGROUND: Network approach has been applied to a wide variety of psychiatric disorders. The aim of the present study was to identify network structures of remitters and non-remitters in patients with first-episode psychosis (FEP) at baseline and the 6-month follow-up. METHODS: Participants (n = 252) from the Korean Early Psychosis Study (KEPS) were enrolled. They were classified as remitters or non-remitters using Andreasen's criteria. We estimated network structure with 10 symptoms (three symptoms from the Positive and Negative Syndrome Scale, one depressive symptom, and six symptoms related to schema and rumination) as nodes using a Gaussian graphical model. Global and local network metrics were compared within and between the networks over time. RESULTS: Global network metrics did not differ between the remitters and non-remitters at baseline or 6 months. However, the network structure and nodal strengths associated with positive-self and positive-others scores changed significantly in the remitters over time. Unique central symptoms for remitters and non-remitters were cognitive brooding and negative-self, respectively. The correlation stability coefficients for nodal strength were within the acceptable range. CONCLUSION: Our findings indicate that network structure and some nodal strengths were more flexible in remitters. Negative-self could be an important target for therapeutic intervention.

Effects of social defeat stress on dopamine D2 receptor isoforms and proteins involved in intracellular trafficking.

BACKGROUND: Chronic social defeat stress induces depression and anxiety-like behaviors in rodents and also responsible for differentiating defeated animals into stress susceptible and resilient groups. The present study investigated the effects of social defeat stress on a variety of behavioral parameters like social behavior, spatial learning and memory and anxiety like behaviors. Additionally, the levels of various dopaminergic markers, including the long and short form of the D2 receptor, and total and phosphorylated dopamine and cyclic adenosine 3',5'-monophosphate regulated phosphoprotein-32, and proteins involved in intracellular trafficking were assessed in several key brain regions in young adult mice. METHODS: Mouse model of chronic social defeat was established by resident-intruder paradigm, and to evaluate the effect of chronic social defeat, mice were subjected to behavioral tests like spontaneous locomotor activity, elevated plus maze (EPM), social interaction and Morris water maze tests. RESULTS: Mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. The susceptible group exhibited greater decreases in time spent in the open and closed arms compared to the control group on the EPM. In the social interaction test, the susceptible group showed greater increases in submissive and neutral behaviors and greater decreases in social behaviors relative to baseline compared to the control group. Furthermore, increased expression of D2L, D2S, Rab4, and G protein-coupled receptor associated sorting protein-1 was observed in the amygdala of the susceptible group compared to the control group. CONCLUSION: These findings suggest that social defeat stress induce anxiety-like and altered social interacting behaviors, and changes in dopaminergic markers and intracellular trafficking-related proteins.

Neuronal signatures of anger and fear in patients with psychosis.

The present study investigated the functional neuroanatomy in response to sentence stimuli related to anger-provoking situations and fear of negative evaluation in patients with psychosis. The tasks consisted of four active conditions, Self-Anger (SA), Self-Fear, Other-Anger (OA), and Other-Fear (OF), and two neutral conditions, Neutral-Anger (NA) and Neutral-Fear (NF). Several relevant clinical measures were obtained. Under all contrasts, significantly higher activation in the left inferior parietal gyrus or superior parietal gyrus and the left middle occipital gyrus or superior occipital gyrus was observed in patients compared to healthy controls (HCs). However, we observed significantly lower activation in the left angular gyrus (AG) and left middle temporal gyrus (MTG) under the OA vs. NA contrast, as well as in the left precuneus and left posterior cingulate gyrus (PCG) under the OF vs. NF contrast in patients. The mean beta values for the significant regions under the SA vs. NA and OF vs. NF contrasts were significantly associated with the total PI and PANSS scores, respectively. These findings indicate that patients with psychosis exhibit hypoactivation in the AG, MTG, precuneus, and PCG compared to HCs. The findings suggest that patients with psychosis are less efficient at recruiting neural responses in those regions for semantic processing and social evaluation.

Altered Neuroanatomical Signatures of Patients With Treatment-Resistant Schizophrenia Compared to Patients With Early-Stage Schizophrenia and Healthy Controls.

Background: The relationship between brain structural changes and cognitive dysfunction in schizophrenia is strong. However, few studies have investigated both neuroanatomical abnormalities and cognitive dysfunction in treatment-resistant schizophrenia (TRS). We examined neuroanatomical markers and cognitive function between patients with TRS or early-stage schizophrenia (ES-S) and healthy controls (HCs). Relationships between neuroanatomical markers and cognitive function in the patient groups were also investigated. Methods: A total of 46 and 45 patients with TRS and ES-S and 61 HCs underwent structural magnetic resonance imaging (MRI) brain scanning and comprehensive cognitive tests. MRI scans were analyzed using the FreeSurfer to investigate differences in cortical surface area (CSA), cortical thickness (CT), cortical volume (CV), and subcortical volume (SCV) among the groups. Four cognitive domains (attention, verbal memory, executive function, and language) were assessed. Comparisons of neuroanatomical and cognitive function results among the three groups were performed. Results: A widespread reduction in CT was observed in patients with TRS compared to HCs, but differences in cortical thinning between TRS and ES-S patients were mainly limited to the inferior frontal gyrus and insula. Several subcortical structures (accumbens, amygdala, hippocampus, putamen, thalamus and ventricles) were significantly altered in TRS patients compared to both ES-S patients and HCs. Performance in the verbal memory domain was significantly worse in TRS patients compared to ES-S patients. A positive relationship between the thickness of the left middle temporal gyrus and the composite score for language was identified in patients with ES-S. Conclusions: Our findings suggest significant cognitive impairment and reductions in CT and SCV in individuals with TRS compared to those with ES-S and HCs. These abnormalities could act as biomarkers for earlier identification of TRS.

Altered amygdala-based functional connectivity in individuals with attenuated psychosis syndrome and first-episode schizophrenia.

Altered resting-state functional connectivity (FC) of the amygdala (AMY) has been demonstrated to be implicated in schizophrenia (SZ) and attenuated psychosis syndrome (APS). Specifically, no prior work has investigated FC in individuals with APS using subregions of the AMY as seed regions of interest. The present study examined AMY subregion-based FC in individuals with APS and first-episode schizophrenia (FES) and healthy controls (HCs). The resting state FC maps of the three AMY subregions were computed and compared across the three groups. Correlation analysis was also performed to examine the relationship between the Z-values of regions showing significant group differences and symptom rating scores. Individuals with APS showed hyperconnectivity between the right centromedial AMY (CMA) and left frontal pole cortex (FPC) and between the laterobasal AMY and brain stem and right inferior lateral occipital cortex compared to HCs. Patients with FES showed hyperconnectivity between the right superficial AMY and left occipital pole cortex and between the left CMA and left thalamus compared to the APS and HCs respectively. A negative relationship was observed between the connectivity strength of the CMA with the FPC and negative-others score of the Brief Core Schema Scales in the APS group. We observed different altered FC with subregions of the AMY in individuals with APS and FES compared to HCs. These results shed light on the pathogenetic mechanisms underpinning the development of APS and SZ.

Effects of antipsychotics on rumination in patients with first-episode psychosis.

BACKGROUND: Rumination is a well-known risk factor for depression. It is also associated with negative and positive symptoms and suicidality in patients suffering from psychosis. However, no studies have addressed the effect of antipsychotics on rumination. METHODS: Using the Brooding Scale (BS), we investigated the effect of antipsychotics on rumination at the 6-month follow up in patients with first-episode psychosis (n = 257). The relationship between rumination and other clinical variables was explored by conducting a correlation analysis and structural equation modeling (SEM). The clinical characteristics and short-term outcomes were compared between high and low ruminators at 6 months. RESULTS: Significant reductions in rumination and various clinical variables were observed at the 6-month follow-up. A significant correlation was observed between rumination and the score on the positive subscale of the Positive and Negative Syndrome Scale (PANSS). A direct path between the PANSS score and rumination was identified by SEM. High ruminators had more severe psychopathology, experienced more childhood traumas, and took less exercise than low ruminators. The recovery rate at 6 months was higher in low ruminators than in high ruminators. CONCLUSIONS: Our findings suggest that antipsychotics are beneficial for reducing rumination in patients with first-episode psychosis. The outcomes at the 6-month follow-up were better in low ruminators than high ruminators.

The Development of the Brooding Scale.

OBJECTIVE: The purpose of this study was to develop a Brooding Scale (BS) and to confirm its psychometric properties. METHODS: A preliminary questionnaire was developed based on a literature review and face-to-face interviews with healthy subjects. To evaluate reliability and construct validity, a 15-item BS was administered to 124 healthy subjects. Convergent validity was tested by assessing the relationship between the BS and the Ruminative Response Scale (RRS). Discriminant validity was confirmed in 58 patients with schizophrenia. RESULTS: The internal consistency for the BS was excellent. An exploratory factor analysis yielded two factors: the emotional (six items) and cognitive (five items) domains, which explained 33.83% and 23.69% of the variance, respectively. The BS total score and scores for factors 1 and 2 showed significant positive correlations with the RRS. The total score and sub-factor scores of the BS were significantly higher in patients with schizophrenia than in healthy subjects. CONCLUSION: The BS can be used as a reliable and valid tool to assess brooding in healthy adults. In addition, it had good discriminant validity for patients with schizophrenia.

Metabolite signature associated with stress susceptibility in socially defeated mice.

OBJECTIVE: Social defeat represents a naturalistic form of conditioned fear and is often used as an animal model of depression. The present study aimed to identify the neurochemicals in select brain regions of mice exposed to social defeat stress. METHODS: Adult C57BL/6N mice were subjected social defeat stress for 10 days. Using high-resolution magic angle spinning 1H nuclear magnetic resonance (HR-MAS 1H NMR), untargeted metabolomes were measured in the amygdala (AMY), dorsal hippocampus (dHIP), dorsal striatum (dST), and prefrontal cortex (PFC). RESULTS: We observed perturbations of glutamine in the AMY; glutamate in the dHIP; glycine and myo-inositol in the dST; and aspartate, choline, and phosphoethanolamine in the PFC of susceptible and/or unsusceptible groups compared to the control group. The susceptible and unsusceptible groups significantly differed with regard to three metabolites: glutamine, glycine, and choline. CONCLUSION: These findings suggest that social defeat stress induces disturbances in the metabolism of amino acids, lipids, and neurotransmitters in several brain areas. The resulting susceptibility-related metabolites may provide new insights into the pathophysiology underlying stress-related mental illness.

Associations of fatty acids with cognition, psychopathology, and brain-derived neurotrophic factor levels in patients with first-episode schizophrenia and related disorders treated with paliperidone extended release.

This study assessed fatty acid and brain-derived neurotrophic factor levels in patients with first-episode schizophrenia and related disorders. The levels of erythrocyte fatty acids and plasma brain-derived neurotrophic factor were measured at baseline and week 8 after treatment with paliperidone extended release. Cognitive function was evaluated using the Cognitive Assessment Interview and the cognition subscale of the Neuroleptic-Induced Deficit Syndrome Scale. There were significant decreases in stearic acid and nervonic acid levels and a significant increase in eicosapentaenoic acid levels after eight weeks. At week 8, cognition was positively associated with dihomo-γ-linolenic acid, linoleic acid, and eicosapentaenoic acid levels, and negatively associated with nervonic acid levels. Psychopathology was positively correlated with polyunsaturated fatty acid levels, and negatively correlated with saturated fatty acid levels at week 8. At both baseline and week 8, brain-derived neurotrophic factor level had a negative association with polyunsaturated fatty acids and a positive association with saturated fatty acids and monounsaturated fatty acids. The present study demonstrated that fatty acids have significant associations with cognition and psychopathology at week 8, and with brain-derived neurotrophic factor levels at both baseline and week 8.

Early predictors of a clinical response at 8 weeks in patients with first-episode psychosis treated with paliperidone ER.

Identification of early clinical markers that predict later treatment outcomes in first-episode psychosis is highly valuable. The present study was conducted to determine the best time at which to predict the late treatment response in first-episode psychosis patients treated with paliperidone extended release (ER), the factors predicting early treatment responses (at Week 2 and Week 3) and the relationships between the paliperidone ER plasma concentrations at Week 2 and Week 3, and the treatment responses at Week 2, Week 3 and Week 8. Various criteria for assessing treatment response were employed. We determined the plasma paliperidone concentrations at Week 2 and Week 3, using validated high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The treatment response at Week 3 optimally predicted the later (Week 8) response, in terms of negative predictive value (NPV). Independent predictors for good treatment responses at Week 2 and Week 3 were: Female gender, a higher educational level, a higher Positive and Negative Syndrome Scale (PANSS) excited score, and/or a shorter duration of untreated psychosis (DUP). The plasma paliperidone concentration at Week 3, but not Week 2, was a significant predictor of the late treatment response at Week 8. These results may help appropriate clinical decision-making for early non-responders after having their first episode of psychosis.

Predictors of suicidal ideation in a community sample: roles of anger, self-esteem, and depression.

The objective of this cross-sectional study was to investigate the relationships of anger, self-esteem, and depression with suicidal ideation. A survey was conducted in a wide range of community areas across Jeollabuk-do Province, Korea. A total of 2964 subjects (mean age=44.4yr) participated in this study. Hierarchical regression was used to investigate predictors of suicidal ideation in terms of their sociodemographic characteristics, depression, self-esteem, and anger. Hierarchical regression analyses revealed that anger and self-esteem were significantly associated with suicidal ideation regardless of age and after controlling for depression. Moderation analysis showed that the impact of anger on suicidal ideation was significantly greater among females than males in adolescents, but not in other age groups. Additionally, there were some differences in sociodemographic predictors of suicidal ideation among age groups. Predictors included gender and family harmony in adolescents, marital status and family harmony in middle-aged individuals, and economic status and family harmony in elderly individuals. Our results revealed that anger and self-esteem play important roles in suicidal ideation beyond the effect of depression. Development and implementation of preventive strategies, including management of anger and self-esteem, could possibly reduce suicidal ideation and subsequent suicide attempts.

Clinical characteristics of patients who have recovered from schizophrenia: the role of empathy and positive-self schema.

AIM: This article compares the socio-demographic and clinical characteristics of patients with schizophrenia who recovered with those who achieved remission. METHODS: Participants were classified based on predetermined criteria for recovery and remission. Data on demographic characteristics, information on duration of untreated psychosis, and assessments of current and historical symptom profiles and socio-occupational functioning emerged from careful chart review and direct interviews. Cross-sectional assessments of clinical variables were derived from the Positive and Negative Syndrome Scale, the Scale for the Assessment of Negative Symptoms, the Personal and Social Performance Scale, the Social Functioning Questionnaire, the Schizophrenia Cognition Rating Scale (ScoRS), the Basic Empathy Scale, and the Brief Core Schema Scales (BCSS). RESULTS: We found no significant differences between recovered and remitted groups with respect to demographic variables or duration of untreated psychosis. Cognitive and total empathy scores, positive-self schema score on the BCSS, and global score on the ScoRS were significantly higher in the recovered than the remitted group. Furthermore, patients with good levels of empathy and positive-self schema and intact neurocognitive functioning were more likely to achieve recovery. CONCLUSION: These results suggest that empathy, positive-self schema and neurocognitive functioning may serve as important clinical characteristics distinguishing those patients who have recovered from those who have achieved only remission.

Efficacy and safety of ziprasidone in the treatment of first-episode psychosis: an 8-week, open-label, multicenter trial.

The aim of this study was to investigate the efficacy and safety of ziprasidone in first-episode psychosis. This was an 8-week, open-label, multicenter trial. In total, 27 patients (14 male patients, 13 female patients) with a Diagnostic and Statistical Manual of Mental Disorders--Fourth Edition diagnosis of schizophreniform disorder, schizoaffective disorder, schizophrenia, or psychotic disorder not otherwise specified comprised the study population. The initial recommended dose of ziprasidone was 40 mg/day. Within the first 2 weeks, the dose could be increased to 120-160 mg/day depending on the patient's condition. The primary outcome variables were scores on the Positive and Negative Syndrome Scale (PANSS) and the Clinical Global Impression-Severity scale; secondary measures included the Calgary Depression Scale for Schizophrenia and others. To assess safety, we measured drug-related adverse events, weight, lipid variables, prolactin, and corrected QT (QTc) interval. Among the 27 enrolled participants, 16 dropped out [lack of efficacy (n = 7), loss to follow-up (n = 7), withdrawn consent (n = 1), and serious adverse event (n = 1)]. The mean total daily and endpoint doses of ziprasidone were 120.30 ± 40.34 and 131.85 ± 51.22 mg/day, respectively. The administration of ziprasidone resulted in significant improvement in the PANSS (P < 0.0001) and CGI scores (P < 0.0001) over time. Significant improvement in the Calgary Depression Scale for Schizophrenia score (P < 0.0001) was also observed at week 8. The response rate (defined as a 30% or greater decrease in the PANSS total score from baseline to last observation) was 51.85%. No significant differences in extrapyramidal symptoms rating scale scores, and lipid and prolactin levels from baseline to last observation were found. However, modest side effects regarding the incidence of extrapyramidal symptoms and hyperprolactinemia, and weight change from baseline in male patients were observed. These results indicate that ziprasidone is effective in the treatment of the positive, negative, and depressive symptoms of first-episode psychosis and has a modest side-effect burden.

No negative symptoms in healthy volunteers after single doses of amisulpride, aripiprazole, and haloperidol: a double-blind placebo-controlled trial.

Noncompliance and poor outcome in patients with schizophrenia are closely related to the negative symptoms secondary to antipsychotics. No controlled study has evaluated whether amisulpride and aripiprazole induce negative symptoms. The aim of this study was to assess the effects of single doses of amisulpride, aripiprazole, haloperidol, and risperidone in healthy volunteers. Seventy-eight young volunteers took part in this double-blind, randomized, placebo-controlled, parallel study of four antipsychotics: 400 mg amisulpride, 10 mg aripiprazole, 3 mg haloperidol, and 2 mg risperidone. Assessments of negative symptoms were done 4 h after administration using both subjective rating scales (Neuroleptic Induced Deficit Syndrome Scale and Subjective Deficit Syndrome Scale) and an objective rating scale (Scale for the Assessment of Negative Symptoms). Risperidone only produced significant increases on the avolition score of the Neuroleptic Induced Deficit Syndrome Scale and blunted affect and alogia scores of the Scale for the Assessment of Negative Symptoms compared with placebo. The effect on blunted affect persisted after controlling for mental sedation. Amisulpride, aripiprazole, and haloperidol did not induce negative symptoms. Aripiprazole and risperidone induced mild extrapyramidal symptoms. The most common adverse events were somnolence and cognitive slowing. These data indicate that a single risperidone dose induces negative symptoms in normal volunteers, whereas amisulpride, aripiprazole, and haloperidol do not. These characteristics of antipsychotics should be considered when choosing optimal drugs for patients with psychosis.

Age-specific effects of 6-hydroxydopamine lesions of the rat medial prefrontal cortex on stress-induced c-fos expression in subcortical areas.

As adolescence is a critical period when dopaminergic neuronal maturation peaks, we hypothesized that 6-hydroxydopamine (OHDA) lesions of the medial prefrontal cortex (mPFC) in adolescent rats would have more negative effects than lesions in adult rats. Therefore, we investigated the effects of 6-OHDA lesions of the mPFC in adolescent and adult rats on stress-induced c-fos expression in the brain. Adolescent and adult Sprague-Dawley rats, aged 4 and 7 weeks on arrival, respectively, were studied. 6-OHDA (8.0 microg) for the lesion groups and ascorbic acid for the sham groups were injected bilaterally into the mPFC. All animals were pretreated with desipramine 30 min before being anesthetized. The control group did not undergo any surgery-related procedure except the desipramine injection. After recovery for 1 week, the rats were subjected to restraint stress for 1 h. Immediately after the stress, the rats were killed and c-fos immunohistochemistry was examined. The c-fos expression in the nucleus accumbens core (AcbC), nucleus accumbens shell (AcbSh), CA1, CA3, dentate gyrus (DG), central amygdaloid (Ce), basolateral amygdaloid (BL), and temporal cortex (Tc) was compared. Adolescent rats with 6-OHDA lesions subjected to restraint stress had greater c-fos expression in the AcbC, AcbSh, DG, Ce, BL, and Tc, compared to the sham and control groups, whereas these differences were not observed among the adult groups. These results suggest that a hypodopaminergic state in the mPFC of adolescent rats, but not adult rats, is related to increased sensitivity to stress, suggesting that damage to or maldevelopment of dopaminergic neurons during adolescence has an age-specific effect. Further research is warranted to investigate the mechanism of the age-specific effect of 6-OHDA lesions of the mPFC.

Clozapine-induced tardive dyskinesia in schizophrenic patients taking clozapine as a first-line antipsychotic drug.

OBJECTIVE: Clozapine causes few extrapyramidal symptoms and is recommended as a treatment drug for severe tardive dyskinesia (TD). However, several case reports have suggested that clozapine could also cause TD. We investigated whether clozapine used as a first-line antipsychotic drug can cause TD. METHOD: We identified 101 patients at Yanbian Socio-Mental Hospital and Yanbian Brain Hospital in China who had received clozapine as a primary antipsychotic drug since their first episode of illness and evaluated the prevalence rate, type, and severity of TD using the Extrapyramidal Symptoms Rating Scale (ESRS). The criterion for TD was a score of > or = 3 on one item or 2 on two or more items of the ESRS. RESULTS: The mean age and duration of illness of the patients were 38.93+/-8.36 and 12.88+/-6.90 years, respectively. The mean duration of clozapine treatment was 12.10+/-6.26 years. The prevalence of TD was 3.96% (4/101). Compared to patients without TD, patients with TD had a long duration of illness and clozapine treatment; all had the orolingual type of TD. TD was relatively mild, with a mean score of 4.75, and tended to accentuate with an activation procedure of rapid pronation and supination of the hands. CONCLUSIONS: These results suggest that clozapine may cause TD; however, the prevalence is low and the severity is relatively mild, with no or mild self-reported discomfort. Therefore, we recommend that regular examination for TD using the activation procedure should be performed in patients who use clozapine on a long-term basis.