Understanding the therapeutic action of antipsychotics: from molecular to cellular targets with focus on the islands of Calleja.

The understanding of the pathophysiology of schizophrenia as well as the mechanisms of action of antipsychotic drugs remains a challenge for psychiatry. The demonstration of the therapeutic efficacy of several new atypical drugs targeting multiple different receptors apart from the classical dopamine D2 receptor as initially postulated unique antipsychotic target, complicated even more conceptualization efforts. Here we discuss results suggesting a main role of the islands of Calleja, still poorly studied GABAergic granule cell clusters in the ventral striatum, as cellular targets of several innovative atypical antipsychotics (clozapine, cariprazine and xanomeline/emraclidine) effective in treating also negative symptoms of schizophrenia. We will emphasize the potential role of dopamine D3 and M4 muscarinic acetylcholine receptor expressed at highest level by the islands of Calleja, as well as their involvement in schizophrenia-associated neurocircuitries. Finally, we will discuss the implications of new data showing ongoing adult neurogenesis of the islands of Calleja, as a very promising antipsychotic target linking long-life neurodevelopment and dopaminergic dysfunction in the striatum.

A systematic scoping review of rodent models of catatonia: Clinical correlations, translation and future approaches.

Catatonia is a psychiatric disorder, which subsumes a plethora of affective, motor and behavioral symptoms. In the last two decades, the number of behavioral and neuroimaging studies on catatonia has steadily increased. The majority of behavioral and neuroimaging studies in psychiatric patients suggested aberrant higher-order frontoparietal networks which, on the biochemical level, are insufficiently modulated by gamma-aminobutyric acid (GABA)-ergic and glutamatergic transmission. However, the pathomechanisms of catatonic symptoms have rarely been studied using rodent models. Here, we performed a scoping review of literature available on PubMed for studies on rodent models of catatonia. We sought to identify what we could learn from pre-clinical animal models of catatonia-like symptoms, their underlying neuronal correlates, and the complex molecular (i.e. genes and neurotransmitter) mechanisms by which its modulation exerts its effects. What becomes evident is that although many transgenic models present catatonia-like symptoms, they have not been used to better understand the pathophysiological mechanisms underlying catatonia so far. However, the identified neuronal correlates of catatonia-like symptoms correlate to a great extent with findings from neuroscience research in psychiatric patients. This points us towards fundamental cortical-striatal-thalamocortical and associated networks modulated by white matter inflammation as well as aberrant dopaminergic, GABAergic, and glutamatergic neurotransmission that is involved in catatonia. Therefore, this scoping review opens up the possibility of finally using transgenic models to help with identifying novel target mechanisms for the development of new drugs for the treatment of catatonia.

Olanzapine Effects on Parvalbumin/GAD67 Cell Numbers in Layers/Subregions of Dorsal Hippocampus of Chronically Socially Isolated Rats.

Depression is linked to changes in GABAergic inhibitory neurons, especially parvalbumin (PV) interneurons, which are susceptible to redox dysregulation. Olanzapine (Olz) is an atypical antipsychotic whose mode of action remains unclear. We determined the effect of Olz on PV-positive (+) and glutamate decarboxylase 67 (GAD67) + cell numbers in the layers of dorsal hippocampus (dHIPP) cornu ammonis (CA1-CA3) and dentate gyrus (DG) subregions in rats exposed to chronic social isolation (CSIS), which is an animal model of depression. Antioxidative enzymes and proinflammatory cytokine levels were also examined. CSIS decreased the PV+ cell numbers in the Stratum Oriens (SO) and Stratum Pyramidale (SP) of dCA1 and dDG. It increased interleukin-6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), and copper-zinc superoxide dismutase (CuZnSOD) levels, and it decreased catalase (CAT) protein levels. Olz in CSIS increased the number of GAD67+ cells in the SO and SP layers of dCA1 with no effect on PV+ cells. It reduced the PV+ and GAD67+ cell numbers in the Stratum Radiatum of dCA3 in CSIS. Olz antagonizes the CSIS-induced increase in CuZnSOD, CAT and SOCS3 protein levels with no effect on IL-6. Data suggest that the protective Olz effects in CSIS may be mediated by altering the number of PV+ and GAD67+ cells in dHIPP subregional layers.

Psilocybin does not induce the vulnerability marker HSP70 in neurons susceptible to Olney's lesions.

S-ketamine, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and psilocybin, a 5-hydroxy-tryptamine (serotonin) 2A receptor (5-HT2AR) agonist, are reported as effective rapid-acting antidepressants. Both compounds increase glutamate signalling and evoke cortical hyperexcitation. S-ketamine induces neurotoxicity especially in the retrosplenial cortex (Olney's lesions). Whether psilocybin produces similar neurotoxic effects has so far not been investigated. We performed an immunohistochemical whole-brain mapping for heat shock protein 70 (HSP70) in rats treated with psilocybin, S-ketamine, and MK-801. In contrast to S-ketamine- and MK-801-treated animals, we did not detect any HSP70-positive neurons in retrosplenial cortex of rats treated with psilocybin. Our results suggest that psilocybin might be safer for clinical use compared to S-ketamine regarding neuronal damage.

Sex-specific differences in metabolic hormone and adipose tissue dynamics induced by moderate low-carbohydrate and ketogenic diet.

Low-carbohydrates diets are increasingly used to treat obesity and metabolic disorders. A very low-carbohydrate, ketogenic diet is hard to follow and, due to the very high fat content, linked to severe side effects, like hyperlipidemia and atherogenesis. Therefore, a less restrictive, unsaturated fat-based low-carbohydrate diet appears as a promising alternative. Since neither sex differences, nor their effect on specific metabolic hormones and adipose tissue compartments have been investigated thoroughly in these diets, we aimed to analyze their dynamics and metabolic factors in mice. We found a significant sexual dimorphism with decreased body weight and subcutaneous fat only in males on ketogenic diet, while diminished insulin, elevated ghrelin and FGF-21 were present with a differential time course in both sexes. The non-ketogenic moderate low-carbohydrate diet increased body weight and perigonadal fat in females, but induced leptin elevation in males. Both diets enhanced transiently TNFɑ only in males and had no impact on behavior. Altogether, these results reveal complex sex-dependent effect of dietary interventions, indicating unexpectedly females as more prone to unfavorable metabolic effects of low-carbohydrate diets.

Metabolic Fingerprints of Effective Fluoxetine Treatment in the Prefrontal Cortex of Chronically Socially Isolated Rats: Marker Candidates and Predictive Metabolites.

The increasing prevalence of depression requires more effective therapy and the understanding of antidepressants' mode of action. We carried out untargeted metabolomics of the prefrontal cortex of rats exposed to chronic social isolation (CSIS), a rat model of depression, and/or fluoxetine treatment using liquid chromatography-high resolution mass spectrometry. The behavioral phenotype was assessed by the forced swim test. To analyze the metabolomics data, we employed univariate and multivariate analysis and biomarker capacity assessment using the receiver operating characteristic (ROC) curve. We also identified the most predictive biomarkers using a support vector machine with linear kernel (SVM-LK). Upregulated myo-inositol following CSIS may represent a potential marker of depressive phenotype. Effective fluoxetine treatment reversed depressive-like behavior and increased sedoheptulose 7-phosphate, hypotaurine, and acetyl-L-carnitine contents, which were identified as marker candidates for fluoxetine efficacy. ROC analysis revealed 4 significant marker candidates for CSIS group discrimination, and 10 for fluoxetine efficacy. SVM-LK with accuracies of 61.50% or 93.30% identified a panel of 7 or 25 predictive metabolites for depressive-like behavior or fluoxetine effectiveness, respectively. Overall, metabolic fingerprints combined with the ROC curve and SVM-LK may represent a new approach to identifying marker candidates or predictive metabolites for ongoing disease or disease risk and treatment outcome.

Ketogenic diet for mood disorders from animal models to clinical application.

Mood disorders such as major depressive disorder (MDD) and bipolar disorder (BD) are often resistant to current pharmacological treatment. Therefore, various alternative therapeutic approaches including diets are, therefore, under investigation. Ketogenic diet (KD) is effective for treatment-resistant epilepsy and metabolic diseases, however, only a few clinical studies suggest its beneficial effect also for mental disorders. Animal models are a useful tool to uncover the underlying mechanisms of therapeutic effects. Women have a twice-higher prevalence of mood disorders but very little is known about sex differences in nutritional psychiatry. In this review, we aim to summarize current knowledge of the sex-specific effects of KD in mood disorders. Ketone bodies improve mitochondrial functions and suppress oxidative stress, inducing neuroprotective and anti-inflammatory effects which are both beneficial for mental health. Limited data also suggest KD-induced improvement of monoaminergic circuits and hypothalamus-pituitary-adrenal axis-the key pathophysiological pathways of mood disorders. Gut microbiome is an important mediator of the beneficial and detrimental effects of diet on brain functioning and mental health. Gut microbiota composition is affected in mood disorders but its role in the therapeutic effects of different diets, including KD, remains poorly understood. Still little is known about sex differences in the effects of KD on mental health as well as on metabolism and body weight. Some animal studies used both sexes but did not find differences in behavior, body weight loss or gut microbiota composition. More studies, both on a preclinical and clinical level, are needed to better understand sex-specific effects of KD on mental health.

Fluoxetine Enhances Synaptic Vesicle Trafficking and Energy Metabolism in the Hippocampus of Socially Isolated Rats.

Chronic social isolation (CSIS)-induced alternation in synaptic and mitochondrial function of specific brain regions is associated with major depressive disorder (MDD). Despite the wide number of available medications, treating MDD remains an important challenge. Although fluoxetine (Flx) is the most frequently prescribed antidepressant, its mode of action is still unknown. To delineate affected molecular pathways of depressive-like behavior and identify potential targets upon Flx treatment, we performed a comparative proteomic analysis of hippocampal purified synaptic terminals (synaptosomes) of rats exposed to six weeks of CSIS, an animal model of depression, and/or followed by Flx treatment (lasting three weeks of six-week CSIS) to explore synaptic protein profile changes. Results showed that Flx in controls mainly induced decreased expression of proteins involved in energy metabolism and the redox system. CSIS led to increased expression of proteins that mainly participate in Ca2+/calmodulin-dependent protein kinase II (Camk2)-related neurotransmission, vesicle transport, and ubiquitination. Flx treatment of CSIS rats predominantly increased expression of proteins involved in synaptic vesicle trafficking (exocytosis and endocytosis), and energy metabolism (glycolytic and mitochondrial respiration). Overall, these Flx-regulated changes in synaptic and mitochondrial proteins of CSIS rats might be critical targets for new therapeutic development for the treatment of MDD.

Region-Specific Enhancement of c-fos Expression by Combined Treatment With NMDA Receptor Agonists and Antagonists With Antidepressant Potential.

Rapastinel, formerly Glyx-13, is a novel positive allosteric modulator of the N-methyl-D-aspartate-receptor (NMDAR) that counteracts psychotomimetic actions of NMDAR antagonists. We set out to evaluate the effect of rapastinel alone or in combination with the global and GluN2B subunit-specific NMDAR antagonists MK-801 and Ro25-6981, respectively, on neuronal activation in relevant regions using c-fos brain mapping. Whereas rapastinel alone did not trigger significant c-fos expression beyond the prelimbic cortex, it strongly increased the c-fos expression induced by MK-801 in hippocampal, cingulate, and retrosplenial areas. Similar results were obtained when rapastinel was replaced by D-cycloserine. Our results reveal new interactions at network level between NMDAR modulators with possible implications regarding their therapeutic effects.

Comparative Severity Assessment of Genetic, Stress-Based, and Pharmacological Mouse Models of Depression.

The use of animals in neurosciences is pivotal to gaining insights into complex functions and dysfunctions of behavior. For example, various forms of physical and/or psychological stress are inherent to various animal models for psychiatric disorders, e.g., depression. Regarding animal welfare, it would be mandatory to use models that inflict the least amount of stress necessary to address the underlying scientific question. This study compared the severity of different approaches to induce depression in mice: mutagenesis in GluA1 knockout, immobilization stress, and stress-induction via stress hormone treatment. While genetic alterations potentially represent a lifelong burden, the temporary intervention only affects the animals for a limited time. Therefore, we used home cage-based behavioral and physiological parameters, including nest building, burrowing, body weight, and fecal corticosterone metabolites, to determine the well-being of male and female mice. In addition, we performed an evidence-based estimate of severity using a composite score for relative severity assessment (RELSA) with this data. We found that even though restraint stress and supplementation of corticosterone in the diet both aimed at depression-related precipitating stress effects, the latter affected the well-being much stronger, especially in females. Restraint leads to less noticeable well-being impairments but causes depression-associated anhedonic behavior. Mice of both sexes recovered well from the stress treatment. GluA1 KO and their littermates showed diminished well-being, comparable to the immobilization experiments. However, since this is a lifelong condition, this burden is not reversible and potentially accumulative. In line with the 3Rs (Replacement, Reduction, and Refinement), the process of choosing the most suitable model should ideally include an evidence-based severity assessment to be able to opt for the least severe alternative, which still induces the desired effect. Promoting refinement, in our study, this would be the restraint stress.

[Rapid-acting antidepressants-neurobiological mechanisms of action]. / Schnell wirksame Antidepressiva ­ neurobiologische Wirkprinzipien.

Rapid-acting antidepressants disprove the dogma that antidepressants need several weeks to become clinically effective. Ketamine, the prototype of a rapid-acting antidepressant, is an N­methyl-D-aspartate (NMDA) receptor blocking agent. A single i.v. application of ketamine induces rapid changes in glutamatergic neurotransmitter systems, leading to preferential activation of glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This evokes the activation of brain-derived neurotrophic factor (BDNF), causing plastic changes in the central nervous system within 24 h. In the prefrontal cortex ketamine leads to a regeneration of synaptic contacts, which have been damaged by chronic stress. This regeneration correlates with improvement of depression-like behavioral changes in rodent models. Classical monoaminergic antidepressants can cause similar changes but with considerably longer latency periods. For clinical application a nasal spray of esketamine has been developed, since this enantiomer has the highest affinity for NMDA receptors; however, since R­ketamine and certain ketamine metabolites also have antidepressant effects in preclinical models, these are currently being tested in clinical studies. Moreover, there are many other glutamatergic substances under clinical investigation for antidepressant effects without ketamine-like adverse effects. In addition, there are also several promising rapid-acting antidepressants that do not primarily act via the glutamate system, such as the gamma-aminobutyric acid (GABA) receptor modulator brexanolone or the serotonin receptor agonist psilocybin.

Cre-Activation in ErbB4-Positive Neurons of Floxed Grin1/NMDA Receptor Mice Is Not Associated With Major Behavioral Impairment.

Extensive evidence suggests a dysfunction of the glutamate NMDA receptor (NMDAR) in schizophrenia, a severe psychiatric disorder with putative early neurodevelopmental origins, but clinical onset mainly during late adolescence. On the other hand, pharmacological models using NMDAR antagonists and the clinical manifestation of anti-NMDAR encephalitis indicate that NMDAR blockade/hypofunction can trigger psychosis also at adult stages, without any early developmental dysfunction. Previous genetic models of NMDAR hypofunction restricted to parvalbumin-positive interneurons indicate the necessity of an early postnatal impairment to trigger schizophrenia-like abnormalities, whereas the cellular substrates of NMDAR-mediated psychosis at adolescent/adult stages are unknown. Neuregulin 1 (NRG1) and its receptor ErbB4 represent schizophrenia-associated susceptibility factors that closely interact with NMDAR. To determine the neuronal populations implicated in "late" NMDAR-driven psychosis, we analyzed the effect of the inducible ablation of NMDARs in ErbB4-expressing cells in mice during late adolescence using a pharmacogenetic approach. Interestingly, the tamoxifen-inducible NMDAR deletion during this late developmental stage did not induce behavioral alterations resembling depression, schizophrenia or anxiety. Our data indicate that post-adolescent NMDAR deletion, even in a wider cell population than parvalbumin-positive interneurons, is also not sufficient to generate behavioral abnormalities resembling psychiatric disorders. Other neuronal substrates that have to be revealed by future studies, may underlie post-adolescent NMDAR-driven psychosis.

Tianeptine modulates synaptic vesicle dynamics and favors synaptic mitochondria processes in socially isolated rats.

Deregulation of synaptic function and neurotransmission has been linked with the development of major depression disorder (MDD). Tianeptine (Tian) has been used as antidepressant with anxiolytic properties and recently as a nootropic to improve cognitive performance, but its mechanism of action is unknown. We conducted a proteomic study on the hippocampal synaptosomal fractions of adult male Wistar rats exposed to chronic social isolation (CSIS, 6 weeks), an animal model of depression and after chronic Tian treatment in controls (nootropic effect) and CSIS-exposed rats (lasting 3 weeks of 6-week CSIS) (therapeutic effect). Increased expression of Syn1 and Camk2-related neurotransmission, vesicle transport and energy processes in Tian-treated controls were found. CSIS led to upregulation of proteins associated with actin cytoskeleton, signaling transduction and glucose metabolism. In CSIS rats, Tian up-regulated proteins involved in mitochondrial energy production, mitochondrial transport and dynamics, antioxidative defense and glutamate clearance, while attenuating the CSIS-increased glycolytic pathway and cytoskeleton organization proteins expression and decreased the expression of proteins involved in V-ATPase and vesicle endocytosis. Our overall findings revealed that synaptic vesicle dynamics, specifically exocytosis, and mitochondria-related energy processes might be key biological pathways modulated by the effective nootropic and antidepressant treatment with Tian and be a potential target for therapeutic efficacy of the stress-related mood disorders.

Correction: Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity.

[This corrects the article DOI: 10.1371/journal.pone.0240439.].

Rapastinel alleviates the neurotoxic effect induced by NMDA receptor blockade in the early postnatal mouse brain.

Rapastinel is a novel psychoactive substance that acts as an N-methyl-D-aspartate-receptor (NMDAR) agonist and triggers antidepressant- and antipsychotic-like effects in animal models. However, it is unknown if rapastinel possesses a better side-effect profile than fast-acting glutamatergic antidepressants, like ketamine, which trigger neurotoxicity in the perinatal rodent cortex and protracted schizophrenia-like alterations. Here we found a remarkable neuroprotective effect of rapastinel against apoptosis induced by the NMDAR antagonist MK-801 in comparison to that elicited by clozapine and the mGlu2/3 agonist LY354740. These results suggest the potential therapeutic/prophylactic effect of rapastinel in ameliorating deleterious effects induced by NMDAR blockade during neurodevelopment.

A neural mechanism for affective well-being: Subgenual cingulate cortex mediates real-life effects of nonexercise activity on energy.

Physical activity substantially improves well-being and mental health, but the underlying brain processes remain unclear. Most research concerns exercise, although the majority of everyday human behaviors, such as walking or stair climbing, are nonexercise activities. Combining neuroimaging with ecological assessment of activity and GPS-triggered smartphone diaries, we show a specific association of nonexercise activity with energy in two independent samples mediated by the subgenual part of the anterior cingulate cortex (sgACC), a key emotion regulatory site. Furthermore, energy predicted a range of mental health metrics. sgACC volume moderated humans' emotional gain from nonexercise activity in real life: Individuals with low sgACC volume, a risk factor for depression, felt less energized when inactive but benefited more from periods of high nonexercise activity. This suggests an everyday life mechanism affecting affective well-being in the general population and, if substantiated in patient samples, a risk and resilience process for mood disorders.

Social isolation in rats: Effects on animal welfare and molecular markers for neuroplasticity.

Early life stress compromises brain development and can contribute to the development of mental illnesses. A common animal model used to study different facets of psychiatric disorders is social isolation from early life on. In rats, this isolation can induce long-lasting alterations in molecular expression and in behavior. Since social isolation models severe psychiatric symptoms, it is to be expected that it affects the overall wellbeing of the animals. As also promoted by the 3Rs principle, though, it is pivotal to decrease the burden of laboratory animals by limiting the number of subjects (reduce, replace) and by improving the animals' wellbeing (refine). The aim of this study was therefore to test possible refinement strategies such as resocialization and mere adult social isolation. We examined whether the alternatives still triggered the necessary phenotype while minimizing the stress load on the animals. Interestingly, we did not find reduced wellbeing-associated burrowing performance in isolated rats. The hyperactive phenotype seen in socially isolated animals was observed for rats undergoing the adult-only isolation, but resocializing ameliorated the locomotor abnormality. Isolation strongly affected markers of neuroplasticity in the prefrontal cortex independent of timing: mRNA levels of Arc, Bdnf and the pool of Bdnf transcripts with the 3' long UTR were reduced in all groups. Bdnf splice variant IV expression was reduced in lifelong-isolated animals. Some of these deficits normalized after resocialization; likewise, exon VI Bdnf mRNA levels were reduced only in animals persistently isolated. Conversely, social deprivation did not affect the expression of Gad67 and Pvb, two GABAergic markers, whereas changes occurred in the expression of dopamine d1 and d2 receptors. As adult isolation was sufficient to trigger the hyperactive phenotype and impaired neuroplasticity in the prefrontal cortex, it could be a candidate for a refinement strategy for certain research questions. To fully grade the severity of post-weaning social isolation and the alternatives, adult isolation and resocialization, a more profound and multimodal assessment approach is necessary.

The impact of handling technique and handling frequency on laboratory mouse welfare is sex-specific.

Handling is a well-known source of stress to laboratory animals and can affect variability of results and even compromise animal welfare. The conventional tail handling in mice has been shown to induce aversion and anxiety-like behaviour. Recent findings demonstrate that the use of alternative handling techniques, e.g. tunnel handling, can mitigate negative handling-induced effects. Here, we show that technique and frequency of handling influence affective behaviour and stress hormone release of subjects in a sex-dependent manner. While frequent tail handling led to a reduction of wellbeing-associated burrowing and increased despair-like behaviour in male mice, females seemed unaffected. Instead, they displayed a stress response to a low handling frequency, which was not detectable in males. This could suggest that in terms of refinement, the impact in handling could differ between the sexes. Independently from this observation, both sexes preferred to interact with the tunnel. Mice generally explored the tunnel more often than the tail-handling hands of the experimenter and showed more positively rated approaches, e.g. touching or climbing, and at the same time, less defensive burrowing, indicating a strong preference for the tunnel.

Fluoxetine modulates neuronal activity in stress-related limbic areas of adult rats subjected to the chronic social isolation.

Antidepressant fluoxetine (Flx) is the first therapeutic choice for the treatment of major depression (MD), however neuroanatomical spots of its action remain unclear. Immunohistochemical detection of c-Fos protein expression has been used for mapping activated neuronal circuits upon various stressors and drugs. We investigated the effect of 3 weeks of Flx treatment (15 mg/kg/day) on changes in neuronal activity, by mapping the number of c-Fos+ cells, in several brain subregions in adult male rats of control and following 3 weeks of chronic social isolation (CSIS), an animal model of depression. The aim was to identify brain subregions activated by vehicle or Flx treatment in both controls or simultaneously applied with CSIS. Flx prevented depressive- and anxiety-like behaviors in CSIS rats. In controls, Flx increased the number of c-Fos+ cells in the anterior/posterior piriform cortex (aPirCx, pPirCx), retrosplenial cortex dysgranular (RSD) and granular, c region (RSGc), dorsal hippocampal subregions (CA1d, CA2, CA3d, DGd), lateral habenula (LHB), paraventricular thalamic nucleus, posterior part (PVP) and lateral/basolateral complex of amygdala (LA/BL). CSIS-induced neuronal activation was observed in brain subregions implicated in mood and other mental disorders such as aPirCx, pPirCx, caudate putamen (CPu), acumbens nucleus shell (AcbSh), RSD, RSGc, DGd, PVP and LA/BL. Flx increased neuronal activation in both controls and CSIS rats in the CA1d, CA2, CA3d, PVP, LA/BL, while in striatum increased neuronal activation was observed only in CSIS. Our data identify activated CSIS-related brain subregions and/or Flx treatment, in which Flx increased c-Fos protein expression in CSIS rats.