Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0-20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.

Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC50) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3-5 mg/kg) and Cmpd-21 (0.4-1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.

Clozapine (CLZ) has been proposed as an agonist for Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to replace Clozapine-N-oxide (CNO); however, there are no reliable guidelines for the use of CLZ for chemogenetic neuromodulation. We titrated the optimal dose of CLZ required to evoke changes in neural activity whilst avoiding off-target effects. We also performed [18F]Fluoro-deoxy-glucose micro positron emission tomography (FDG-microPET) scans to determine the global effect of CLZ-induced hM3D(Gq) DREADD activation in the rat brain. Our results show that low doses of CLZ (0.1 and 0.01 mg/kg) successfully induced neural responses without off-target effects. CLZ at 1 mg/kg evoked a stronger and longer-lasting neural response but produced off-target effects, observed as changes in locomotor behavior and FDG-microPET imaging. Unexpectedly, FDG-microPET imaging failed to demonstrate an increase in regional glucose metabolism in the stimulated cortex during CLZ chemogenetic neuromodulation. Therefore, caution should be used when interpreting FDG-PET images in the context of cortical chemogenetic activation.

The low efficacy of antipsychotic drugs (e.g., clozapine) for negative symptoms and cognitive impairment has led to the introduction of adjuvant therapies. Because previous data suggest the procognitive potential of the antidiabetic drug metformin, this study aimed to assess the effects of chronic clozapine and metformin oral administration (alone and in combination) on locomotor and exploratory activities and cognitive function in a reward-based test in control and a schizophrenia-like animal model (Wisket rats). As impaired dopamine D1 receptor (D1R) function might play a role in the cognitive dysfunctions observed in patients with schizophrenia, the second goal of this study was to determine the brain-region-specific D1R-mediated signaling, ligand binding, and mRNA expression. None of the treatments affected the behavior of the control animals significantly; however, the combination treatment enhanced D1R binding and activation in the cerebral cortex. The Wisket rats exhibited impaired motivation, attention, and cognitive function, as well as a lower level of cortical D1R binding, signaling, and gene expression. Clozapine caused further deterioration of the behavioral parameters, without a significant effect on the D1R system. Metformin blunted the clozapine-induced impairments, and, similarly to that observed in the control animals, increased the functional activity of D1R. This study highlights the beneficial effects of metformin (at the behavioral and cellular levels) in blunting clozapine-induced adverse effects.

Multiple sclerosis is a disease characterised by axonal demyelination in the central nervous system (CNS). The atypical antipsychotic drug clozapine attenuates experimental autoimmune encephalomyelitis (EAE), a mouse model used to study multiple sclerosis, but the precise mechanism is unknown and could include both peripheral and CNS-mediated effects. To better understand where clozapine exerts its protective effects, we investigated the tissue distribution and localisation of clozapine using matrix-assisted laser desorption ionization imaging mass spectrometry and liquid chromatography-mass spectrometry. We found that clozapine was detectable in the brain and enriched in specific brain regions (cortex, thalamus and olfactory bulb), but the distribution was not altered by EAE. Furthermore, although not altered in other organs, clozapine levels were significantly elevated in serum during EAE. Because clozapine antagonises dopamine receptors, we analysed dopamine levels in serum and brain as well as dopamine receptor expression on brain-resident and infiltrating immune cells. While neither clozapine nor EAE significantly affected dopamine levels, we observed a significant downregulation of dopamine receptors 1 and 5 and up-regulation of dopamine receptor 2 on microglia and CD4+-infiltrating T cells during EAE. Together these findings provide insight into how neuroinflammation, as modelled by EAE, alters the distribution and downstream effects of clozapine.

Chronic treatment with second-generation antipsychotic drugs (SGAs) has been associated with an increased risk of metabolic syndrome. To evaluate the longitudinal changes in glucose-lipid homeostasis after SGA use, we studied the time-dependent effects of olanzapine (OLZ) (3 mg/kg, b.i.d.) or clozapine (CLZ) (20 mg/kg, b.i.d.) treatment on metabolic profiles for 9 weeks in rats. Although only OLZ significantly increased body weight in rats, both OLZ and CLZ elevated blood lipid levels. Chronic OLZ treatment induced significant weight gain leading to a higher fasting insulin level and impaired glucose tolerance, whereas CLZ lowered fasting insulin levels and impaired glucose tolerance independent of weight gain. Treatment with both drugs deranged AKT/GSK phosphorylation and up-regulated muscarinic M3 receptors in the rats' livers. Consistent with an elevation in lipid levels, both OLZ and CLZ significantly increased the protein levels of nuclear sterol regulatory element-binding proteins (SREBPs) in the liver, which was associated with improvement in hepatic histamine H1R. However, enhanced carbohydrate response element binding protein (ChREBP) signalling was observed in only CLZ-treated rats. These results suggest that SGA-induced glucose-lipid metabolic disturbances could be independent of weight gain, possibly through activation of SREBP/ChREBP in the liver.

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

Clozapine (CLZ), the sole antipsychotic with superior efficacy for ultra-treatment resistant schizophrenia (TRS), is limited by adverse effects, including metabolic dysregulation. Clozapine's main metabolite, N-desmethylclozapine (NDMC), has potent 5-HT2C antagonist properties which may explain this metabolic dysfunction, thus the CLZ:NDMC ratio is of particular interest. High insulin resistance states could be associated with CYP1A2 induction and lower CLZ:NDMC ratios. Additionally, lower CLZ:NDMC ratios have been associated with better cognitive, but worse metabolic functioning. This study investigated associations between metabolic and cognitive parameters with the CLZ/NDMC ratio. Primary outcomes included relationships between the CLZ:NDMC ratio to the homeostatic model assessment for insulin resistance (HOMA-IR) and Brief Assessment of Cognition in Schizophrenia (BACS) composite z-scores. Secondary outcomes assessed relationships between CLZ:NDMC ratios to fasting insulin, BMI, weight, fasting glucose, and BACS digit sequencing z-scores. 38 patients who were overweight or obese with schizophrenia or schizoaffective disorder completed fasting bloodwork, anthropometric, psychopathological, and cognitive assessments. Multivariate regressions found a statistically significant inverse association between the CLZ/NDMC ratio and HOMA-IR (B = - 1.028, SE B = .473, β = - 0.348 p = 0.037), which may have been driven by fasting insulin levels (B = - 27.124, SE B = 12.081, β = - 0.351 p = 0.031). The CLZ/NDMC ratio may predict insulin resistance/metabolic comorbidity among patients with TRS receiving clozapine.

Schizophrenia is a chronic, severe and disabling psychiatric disorder, whose treatment is based on psychosocial interventions and the use of antipsychotic drugs. While the effects of these drugs are well elucidated in neuronal cells, they are still not so clear in oligodendrocytes, which play a vital role in schizophrenia. Thus, we aimed to characterize biochemical profiles by proteomic analyses of human oligodendrocytes (MO3.13) which were matured using a protocol we developed and treated with either haloperidol (a typical antipsychotic), clozapine (an atypical antipsychotic) or a clozapine + D-serine co-treatment, which has emerged lately as an alternative type of treatment. This was accomplished by employing shotgun proteomics, using nanoESI-LC-MS/MS label-free quantitation. Proteomic analysis revealed biochemical pathways commonly affected by all tested antipsychotics were mainly associated to ubiquitination, proteasome degradation, lipid metabolism and DNA damage repair. Clozapine and haloperidol treatments also affected proteins involved with the actin cytoskeleton and with EIF2 signaling. In turn, metabolic processes, especially the metabolism of nitrogenous compounds, were a predominant target of modulation of clozapine + D-serine treatment. In this context, we seek to contribute to the understanding of the biochemical and molecular mechanisms involved in the action of antipsychotics on oligodendrocytes, along with their possible implications in schizophrenia.

Atypical antipsychotics (AAPs) are considered to possess superior efficacy for treating both the positive and negative symptoms of schizophrenia; however, AAP use often causes excessive weight gain and metabolic abnormalities. Recently, several reports have demonstrated that AAPs activate sterol regulatory element-binding protein (SREBP). SREBP, SREBP cleavage-activating protein (SCAP) and insulin-induced gene (Insig) regulate downstream cholesterol and fatty acid biosynthesis. In this study, we explored the effects of clozapine, olanzapine and risperidone on SREBP signaling and downstream lipid biosynthesis genes in the early events of adipogenic differentiation in adipose-derived stem cells (ASCs). After the induction of adipogenic differentiation for 2 days, all AAPs, notably clozapine treatment for 3 and 7 days, enhanced the expression of SREBP-1 and its downstream lipid biosynthesis genes without dexamethasone and insulin supplementation. Simultaneously, protein level of SREBP-1 was significantly enhanced via inhibition of Insig-2 expression. By contrast, SREBP-1 activation was suppressed when Insig-2 expression was upregulated by transfection with Insig-2 plasmid DNA. In summary, our results indicate that AAP treatment, notably clozapine treatment, induces early-stage lipid biosynthesis in ASCs. Such abnormal lipogenesis can be reversed when Insig-2 expression was increased, suggesting that Insig/SCAP/SREBP signaling may be a therapeutic target for AAP-induced weight gain and metabolic abnormalities.

Pharmacological treatment of mental disorders is currently decided based on "trial and error" strategy. Mitochondrial multifaceted dysfunction is assumed to be a major factor in the pathophysiology and treatment of schizophrenia (SZ) and bipolar disorder (BD). This study aimed to explore the feasibility of using a profile of mitochondrial function parameters as a tool to predict the optimal drug for an individual patient (personalized medicine). Healthy controls (n = 40), SZ (n = 48) and BD (n = 27) patients were recruited. Mental and global state of the subjects, six mitochondrial respiration parameters and 14 mitochondrial function-related proteins were assessed in fresh lymphocytes following in-vitro or in-vivo treatment with five antipsychotic drugs and two mood-stabilizers. In healthy controls, hierarchal clustering shows a drug-specific effect profile on the different mitochondrial parameters following in-vitro exposure. Similar changes were observed in untreated SZ and BD patients with psychosis. Following a month of treatment of the latter patients, only responders showed a significant correlation between drug-induced in-vitro effect (prior to in-vivo treatment) and short-term in-vivo treatment effect for 45% of the parameters. Long- but not short-term psychotropic treatment normalized mitochondria-related parameters in patients with psychosis. Taken together, these data substantiate mitochondria as a target for psychotropic drugs and provide a proof of concept for selective mitochondrial function-related parameters as a predictive tool for an optimized psychotropic treatment in a given patient. This, however, needs to be repeated with an expanded sample size and additional mitochondria related parameters.

Disc1 is a susceptibility gene for psychiatric disorders including schizophrenia. It has been suggested that excess transmission through dopamine type 2 receptors (D2Rs) in the striatum is an underlying mechanism of pathogenesis. In this study, we used super-resolution microscopy to study the distribution of D2Rs at the nanoscale in mice lacking exons 2 and 3 of Disc1 (Disc1-deficient mice). We found that D2Rs in the nucleus accumbens (NAc) of wild-type mice form nanoclusters (~ 20,000 nm2), and that Disc1-deficient mice have larger and more D2R nanoclusters than wild-type mice. Interestingly, administration of clozapine reduced the size and spatial distribution of the nanoclusters only in Disc1-deficient mice. Moreover, we observed that medium spiny neurons in the NAc of Disc1-deficient mice had reduced spine density on their dendrites than did wild-type mice, and this was also reversed by clozapine administration. The altered D2R nanoclusters might be morphological representations of the altered dopaminergic transmission in disease states such as schizophrenia.

Mitochondrial pathology has been implicated in the pathogenesis of psychotic disorders. A few studies have proposed reduced leukocyte mitochondrial DNA (mtDNA) copy number in schizophrenia and bipolar disorder type I, compared to healthy controls. However, it is unknown if mtDNA copy number alteration is driven by psychosis, comorbidity or treatment. Whole blood mtDNA copy number was determined in 594 psychosis patients and corrected for platelet to leukocyte count ratio (mtDNAcnres). The dependence of mtDNAcnres on clinical profile, metabolic comorbidity and antipsychotic drug exposure was assessed. mtDNAcnres was reduced with age (β = -0.210, p < 0.001), use of clozapine (β = -0.110,p = 0.012) and risperidone (β = -0.109,p = 0.014), dependent on prescribed dosage (p = 0.006 and p = 0.026, respectively), and the proportion of life on treatment (p = 0.006). Clozapine (p = 0.0005) and risperidone (p = 0.0126) had a reducing effect on the mtDNA copy number also in stem cell-derived human neurons in vitro at therapeutic plasma levels. For patients not on these drugs, psychosis severity had an effect (β = -0.129, p = 0.017), similar to age (β = -0.159, p = 0.003) and LDL (β = -0.119, p = 0.029) on whole blood mtDNAcnres. Further research is required to determine if mtDNAcnres reflects any psychosis-intrinsic mitochondrial changes.

Antipsychotic drugs target primarily dopaminergic system which makes catechol-O-methyltransferase (COMT) an interesting target in studies searching for treatment response predictors in schizophrenia. The study assessed the association of the COMT rs4680 and rs4818 polymorphisms with therapeutic response to olanzapine, risperidone, clozapine or other antipsychotic medication after 8 weeks of monotherapy in patients with schizophrenia. 521 Caucasian patients with schizophrenia received a monotherapy with olanzapine (10-20 mg/day; N = 190), risperidone (3-6 mg/day; N = 99), or clozapine (100-500 mg/day; N = 102). The fourth group (N = 130) consisted of patients receiving haloperidol (3-15 mg/day), fluphenazine (4-25 mg/day) or quetiapine (50-800 mg/day). Treatment response was defined as a 50% reduction from the baseline positive and negative syndrome scale (PANSS) total and subscale scores, but also as an observed percentage reduction from the initial PANSS0-6 total and subscale scores. Carriers of the COMT rs4680 A allele and carriers of the COMT rs4680-rs4818 C-A haplotype block had greater reduction in the PANSS total scores following olanzapine treatment, compared to carriers of the COMT rs4680 GG genotype and other COMT rs4680-rs4818 haplotypes. The COMT rs4680 A allele, and COMT rs4680-rs4818 C-A haplotype, were significantly associated with therapeutic response in patients treated with olanzapine, but not in patients treated with other antipsychotics.

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization.

Disrupted-in-schizophrenia-1 (DISC1) has emerged as a convincing susceptibility gene for multiple mental disorders, but its mechanistic link to the pathogenesis of schizophrenia related psychiatric conditions is yet to be further understood. Here, we showed that DISC1 localizes to the outer surface of the endoplasmic reticulum (ER). EXOC1, a subunit of the exocyst complex, interacted with DISC1 and affected its recruitment to inositol-1,4,5-trisphosphate receptor 1 (IP3R1). Notably, knockdown of DISC1 and EXOC1 elicited an exaggerated ER calcium response upon stimulation of IP3R agonists. Similar abnormal ER calcium responses were observed in hippocampal neurons from DISC1-deficient mutant mice. Moreover, perturbation of ER calcium dynamics upon DISC1 knockdown was effectively reversed by treatment with antipsychotic drugs, such as clozapine and haloperidol. These results collectively indicate that DISC1 is a regulatory factor in ER calcium dynamics, linking a perturbed intracellular calcium signaling and schizophrenia pathogenesis.

To conduct a systematic review of the comparative efficacy of various psychotropic medications for the treatment of disruptive behavior (DBs) in youths. To this aim, we systematically reviewed randomized clinical trials (RCTs) of various psychotropic medications targeting symptoms of DBs and applied network meta-analysis to investigate their relative efficacy. Fifty-five RCTs meeting the inclusion criteria were selected. To predict and interpret relative treatment efficacy, we compared the efficacy of various psychotropic medications prescribed for DB symptoms based on their mechanism of action. Network meta-analysis revealed that for reducing DBs, second-generation antipsychotics, stimulants, and non-stimulant ADHD medications were more efficacious than placebo, and second-generation antipsychotics were the most efficacious. The dopaminergic modulation of top-down inhibitory process by these medications is discussed in this review. This study offers information on the relative efficacy of various psychotropic medications for the treatment of DB, and insight into a potential neurobiological underpinning for those symptoms. It also illustrates the potential utility of these neurobiological mechanisms as a target for future treatment studies.

G-protein signalling is an evolutionary conserved concept highlighting its fundamental impact on developmental and functional processes. Studies on the effects of G protein signals on tissues as well as an entire organism are often conducted in Caenorhabditis elegans. To understand and control dynamics and kinetics of the processes involved, pharmacological modulation of specific G protein pathways would be advantageous, but is difficult due to a lack in accessibility and regulation. To provide this option, we designed G protein-coupled receptor-based designer receptors (DREADDs) for C. elegans. Initially described in mammalian systems, these modified muscarinic acetylcholine receptors are activated by the inert drug clozapine-N-oxide, but not by their endogenous agonists. We report a novel C. elegans-specific DREADD, functionally expressed and specifically activating Gq-protein signalling in vitro and in vivo which we used for modulating mating behaviour. Therefore, this novel designer receptor demonstrates the possibility to pharmacologically control physiological functions in C. elegans.

The paraventricular thalamic nucleus (PVT) is a part of epithalamus and sends outputs to emotion-related brain areas such as the medial prefrontal cortex, nucleus accumbens, and amygdala. Various functional roles of the PVT in emotion-related behaviors are drawing attention. Here, we investigated the effect of manipulation of PVT neurons on the firing patterns of medial prefrontal cortical (mPFC) neurons and depression-like behavior. Extracellular single-unit recordings revealed that acute activation of PVT neurons by hM3Dq, an activation type of designer receptors exclusively activated by designer drugs (DREADDs), and administration of clozapine N-oxide (CNO) caused firing rate changes in mPFC neurons. Moreover, chronic presynaptic inhibition in PVT neurons by tetanus toxin (TeTX) increased the proportion of interneurons among firing neurons in mPFC and shortened the immobility time in the forced swimming test, whereas long-term activation of PVT neurons by hM3Dq caused recurrent hypoactivity episodes. These findings suggest that PVT neurons regulate the excitation/inhibition balance in the mPFC and mood stability.