Perinatal stress modulates glutamatergic functional connectivity: A post-synaptic density immediate early gene-based network analysis.

Early life stress may induce synaptic changes within brain regions associated with behavioral disorders. Here, we investigated glutamatergic functional connectivity by a postsynaptic density immediate-early gene-based network analysis. Pregnant female Sprague-Dawley rats were randomly divided into two experimental groups: one exposed to stress sessions and the other serving as a stress-free control group. Homer1 expression was evaluated by in situ hybridization technique in eighty-eight brain regions of interest of male rat offspring. Differences between the perinatal stress exposed group (PRS) (n = 5) and the control group (CTR) (n = 5) were assessed by performing the Student's t-test via SPSS 28.0.1.0 with Bonferroni correction. Additionally, all possible pairwise Spearman's correlations were computed as well as correlation matrices and networks for each experimental group were generated via RStudio and Cytoscape. Perinatal stress exposure was associated with Homer1a reduction in several cortical, thalamic, and striatal regions. Furthermore, it was found to affect functional connectivity between: the lateral septal nucleus, the central medial thalamic nucleus, the anterior part of the paraventricular thalamic nucleus, and both retrosplenial granular b cortex and hippocampal regions; the orbitofrontal cortex, amygdaloid nuclei, and hippocampal regions; and lastly, among regions involved in limbic system. Finally, the PRS networks showed a significant reduction in multiple connections for the ventrolateral part of the anteroventral thalamic nucleus after perinatal stress exposure, as well as a decrease in the centrality of ventral anterior thalamic and amygdaloid nuclei suggestive of putative reduced cortical control over these regions. Within the present preclinical setting, perinatal stress exposure is a modifier of glutamatergic early gene-based functional connectivity in neuronal circuits involved in behaviors relevant to model neurodevelopmental disorders.

Dopamine Dynamics and Neurobiology of Non-Response to Antipsychotics, Relevance for Treatment Resistant Schizophrenia: A Systematic Review and Critical Appraisal.

Treatment resistant schizophrenia (TRS) is characterized by a lack of, or suboptimal response to, antipsychotic agents. The biological underpinnings of this clinical condition are still scarcely understood. Since all antipsychotics block dopamine D2 receptors (D2R), dopamine-related mechanisms should be considered the main candidates in the neurobiology of antipsychotic non-response, although other neurotransmitter systems play a role. The aims of this review are: (i) to recapitulate and critically appraise the relevant literature on dopamine-related mechanisms of TRS; (ii) to discuss the methodological limitations of the studies so far conducted and delineate a theoretical framework on dopamine mechanisms of TRS; and (iii) to highlight future perspectives of research and unmet needs. Dopamine-related neurobiological mechanisms of TRS may be multiple and putatively subdivided into three biological points: (1) D2R-related, including increased D2R levels; increased density of D2Rs in the high-affinity state; aberrant D2R dimer or heteromer formation; imbalance between D2R short and long variants; extrastriatal D2Rs; (2) presynaptic dopamine, including low or normal dopamine synthesis and/or release compared to responder patients; and (3) exaggerated postsynaptic D2R-mediated neurotransmission. Future points to be addressed are: (i) a more neurobiologically-oriented phenotypic categorization of TRS; (ii) implementation of neurobiological studies by directly comparing treatment resistant vs. treatment responder patients; (iii) development of a reliable animal model of non-response to antipsychotics.

Disorganization domain as a putative predictor of Treatment Resistant Schizophrenia (TRS) diagnosis: A machine learning approach.

BACKGROUND: Treatment Resistant Schizophrenia (TRS) is the persistence of significant symptoms despite adequate antipsychotic treatment. Although consensus guidelines are available, this condition remains often unrecognized and an average delay of 4-9 years in the initiation of clozapine, the gold standard for the pharmacological treatment of TRS, has been reported. We aimed to determine through a machine learning approach which domain of the Positive and Negative Syndrome Scale (PANSS) 5-factor model was most associated with TRS. METHODS: In a cross-sectional design, 128 schizophrenia patients were classified as TRS (n = 58) or non-TRS (n = 60) after a structured retrospective-prospective analysis of treatment response. The random forest algorithm (RF) was trained to analyze the relationship between the presence/absence of TRS and PANSS-based psychopathological factor scores (positive, negative, disorganization, excitement, and emotional distress). As a complementary strategy to identify the variables most associated with the diagnosis of TRS, we included the variables selected by the RF algorithm in a multivariate logistic regression model. RESULTS: according to the RF model, patients with higher disorganization, positive, and excitement symptom scores were more likely to be classified as TRS. The model showed an accuracy of 67.19%, a sensitivity of 62.07%, and a specificity of 71.43%, with an area under the curve (AUC) of 76.56%. The multivariate model including disorganization, positive, and excitement factors showed that disorganization was the only factor significantly associated with TRS. Therefore, the disorganization factor was the variable most consistently associated with the diagnosis of TRS in our sample.

Relationships between early age at onset of psychotic symptoms and treatment resistant schizophrenia.

AIM: Early age at schizophrenia onset (EOS) has been associated with a worse clinical course, although previous studies reported substantial heterogeneity. Despite the relevance of the subject, the relationship between the age of onset and treatment resistant schizophrenia (TRS) is less clear. METHODS: We screened 197 non-affective psychotic patients. Of these, 99 suffered from schizophrenia and were putative TRS and were included in a prospective 4-to-8-week trial to assess their response to antipsychotics. According to status (TRS/nonTRS) and age-at-onset (early: ≤18 years, EOS; adult: >18 years, adult onset schizophrenia [AOS]) patients were subdivided in EOS-TRS, EOS-nonTRS, AOS-TRS, AOS-nonTRS. Multiple clinical variables were measured and compared by analysis of covariance (ANCOVA), using age as a covariate. Two-way analysis of variance (ANOVA) was used to assess whether significant differences were attributable to TRS status or age-at-onset. RESULTS: The rate of TRS patients was significantly higher in EOS compared to AOS. At the ANCOVA, EOS-TRS had significantly worse clinical, cognitive, and psychosocial outcomes compared to the other groups. Overall, EOS-TRS were more impaired than EOS-nonTRS, while significant differences with AOS-TRS were less consistent, albeit appreciable. Two-way ANOVA demonstrated that, in the majority of the investigated variables, the significant differences among groups were attributable to the TRS status effect rather than to age-at-onset or combined effects. CONCLUSIONS: These results suggest that refractoriness to antipsychotics may be strongly linked to the early onset of psychotic symptoms, possibly as a result of common neurobiology.

Modulation of glutamatergic functional connectivity by a prototypical antipsychotic: Translational inference from a postsynaptic density immediate-early gene-based network analysis.

BACKGROUND: Although extensively studied, the effect of antipsychotics is not completely understood at a network level. We tested the hypothesis that acute administration of haloperidol would modulate functional connectivity of brain regions relevant to schizophrenia pathophysiology. To assess putative changes in brain network properties and regional interactivity, we studied the expression of Homer1a, an Immediate Early Gene (IEG) demonstrated to be induced by antipsychotic administration and coding for a protein involved in glutamatergic synapses remodeling. METHODS: Sprague-Dawley rats (n = 26) assigned to vehicle (VEH; NaCl 0.9%) or haloperidol (HAL; 0.8 mg/kg) were included in the network analysis. Homer1a mRNA induction was evaluated by in situ hybridization. Signal intensity analysis was performed in 33 Regions of Interest (ROIs) in the cortex, the caudate putamen, and the nucleus accumbens. A signal correlation analysis was performed, computing all possible pairwise Pearson correlations among ROIs in the two groups. Two networks were generated for HAL and VEH groups, and their properties and topography were explored. RESULTS: VEH and HAL networks showed qualitative differences in global efficiency and clustering coefficient. The HAL network showed enhanced interactivity between cortical and striatal regions, and within caudate putamen subdivisions. On the other hand, it exhibited reduced inter-correlations between cingulate cortex and anterior insula and caudate putamen and nucleus accumbens. Moreover, haloperidol was able to modulate centrality of crucial functional hubs. These preclinical results corroborate and expand the clinical evidence that antipsychotics may modulate specific brain network properties and disease-related circuits' interactivity.

The Effects of Antipsychotics on the Synaptic Plasticity Gene Homer1a Depend on a Combination of Their Receptor Profile, Dose, Duration of Treatment, and Brain Regions Targeted.

BACKGROUND: Antipsychotic agents modulate key molecules of the postsynaptic density (PSD), including the Homer1a gene, implicated in dendritic spine architecture. How the antipsychotic receptor profile, dose, and duration of administration may influence synaptic plasticity and the Homer1a pattern of expression is yet to be determined. METHODS: In situ hybridization for Homer1a was performed on rat tissue sections from cortical and striatal regions of interest (ROI) after acute or chronic administration of three antipsychotics with divergent receptor profile: Haloperidol, asenapine, and olanzapine. Univariate and multivariate analyses of the effects of topography, treatment, dose, and duration of antipsychotic administration were performed. RESULTS: All acute treatment regimens were found to induce a consistently higher expression of Homer1a compared to chronic ones. Haloperidol increased Homer1a expression compared to olanzapine in striatum at the acute time-point. A dose effect was also observed for acute administration of haloperidol. CONCLUSIONS: Biological effects of antipsychotics on Homer1a varied strongly depending on the combination of their receptor profile, dose, duration of administration, and throughout the different brain regions. These molecular data may have translational valence and may reflect behavioral sensitization/tolerance phenomena observed with prolonged antipsychotics.

Synthesis and Preclinical Evaluation of 6-[18F]Fluorine-α-methyl-l-tryptophan, a Novel PET Tracer for Measuring Tryptophan Uptake.

The positron emission tomography (PET) radioligand α-[11C]methyl-l-tryptophan ([11C]AMT) has been used to assess tryptophan metabolism in cancer, epilepsy, migraine, and autism. Despite its extensive application, the utility of this tracer is currently hampered by the short half-life of the radionuclide used for its labeling (11C, t1/2 = 20.4 min). We herein report the design, synthesis, radiolabeling, and initial in vivo evaluation of a fluorine-18 (18F, t1/2 = 109.7 min) labeled analogue that is fluorinated in the 6-position of the aromatic ring ([18F]6-F-AMTr). In a head-to-head comparison between [18F]6-F-AMTr and [11C]AMT in mice using PET, peak brain radioactivity, regional brain distribution, and kinetic profiles were similar between the two tracers. [18F]6-F-AMTr was however not a substrate for IDO1 or TPH as determined in in vitro enzymatic assays. The brain uptake of the tracer is thus more likely related to LAT1 transport over the blood-brain barrier than metabolism along the serotonin or kynurenine pathways.

Glutamatergic postsynaptic density in early life stress programming: Topographic gene expression of mGlu5 receptors and Homer proteins.

Type-5 metabotropic glutamate receptors (mGlu5) have been implicated in the mechanism of resilience to stress. They form part of the postsynaptic density (PSD), a thickening of the glutamatergic synapse that acts as a multimodal hub for multiple cellular signaling. Perinatal stress in rats triggers alterations that make adult offspring less resilient to stress. In the present study, we examined the expression of gene encoding the mGlu5 (Grm5), as well as those encoding the short and long isoforms of Homer proteins in different brain regions of the offspring of dams exposed to repeated episodes of restraint stress during pregnancy ("perinatally stressed" or PRS offspring). To this end, we investigated unconditioned behavioral response using the light/dark box test, as well as the expression of PSD genes (Homer1a, Homer1b, and Grm5), in the medial prefrontal cortex, cortex, caudate-putamen, amygdala, and dorsal hippocampus. PRS rats spent significantly less time in the light area than the control group. In the amygdala, Homer1a mRNA levels were significantly increased in PRS rats, whereas Homer1b and Grm5 mRNA levels were reduced. In contrast, the transcript encoding for Homer1a was significantly reduced in the medial prefrontal cortex, caudate-putamen, and dorsal hippocampus of PRS rats. We also evaluated the relative ratio between Homer1a and Homer1b/Grm5 expression, finding a significant shift toward the expression of Homer1a in the amygdala and toward Homer1b/Grm5 in the other brain regions. These topographic patterns of Homer1a, Homer1b, and mGlu5 gene expression were significantly correlated with risk-taking behavior measured in the light/dark box test. Remarkably, in the amygdala and in other brain regions, Homer1b and Grm5 expression showed positive correlation with time spent in the light box, whereas Homer1a in the amygdala showed a negative correlation with risk-taking behavior, in contrast with all other brain regions analyzed, wherein these correlations were positive. These results suggest that perinatal stress programs the developmental expression of PSD molecules involved in mGlu5 signaling in discrete brain regions, with a predominant role for the amygdala.

Translating preclinical findings in clinically relevant new antipsychotic targets: focus on the glutamatergic postsynaptic density. Implications for treatment resistant schizophrenia.

There is a growing interest in new molecular targets for antipsychotic therapy. Multiple signal transduction systems have been recently implicated in the pathophysiology of schizophrenia. However, the weight of each specific mechanism remains controversial. A need for a more vigorous approach to the pharmacotherapy of schizophrenia arises from the bedside: about 30-40% of patients do not respond to antipsychotic therapy. Postsynaptic Density (PSD) proteins have recently attracted attention for their role in signal transduction modulation and for their potential implication in psychosis and cognition. The involvement of PSD in the pathophysiology of schizophrenia is supported by post mortem studies, preclinical animal models, modulation by antipsychotics, and association of PSD genes with schizophrenia in GWAS. Taken together, these studies underline the role of PSD modulation, its effects on striatal function and its relationship with motor, executive- and cognitive-like functions suggesting a potential role of PSD proteins as a l target of novel intervention in the treatment of refractory psychosis.

Disease Severity in Treatment Resistant Schizophrenia Patients Is Mainly Affected by Negative Symptoms, Which Mediate the Effects of Cognitive Dysfunctions and Neurological Soft Signs.

This post-hoc study was aimed at assessing whether disease severity was higher in a sample of Treatment Resistant Schizophrenia patients (TRS) compared to schizophrenia patients responsive to antipsychotics (non-TRS). Determinants of disease severity were also investigated in these groups. Eligible patients were screened by standardized diagnostic algorithm to categorize them as TRS or non-TRS. All patients underwent the following assessments: CGI-S; PANSS; DAI; NES; a battery of cognitive tests. Socio-demographic and clinical variables were also recorded. TRS patients exhibited significantly higher disease severity and psychotic symptoms, either as PANSS total score or subscales' scores. A preliminary correlation analysis ruled out clinical and cognitive variables not associated with disease severity in the two groups. Hierarchical linear regression showed that negative symptoms were the clinical variable explaining the highest part of variation in disease severity in TRS, while in non-TRS patients PANSS-General Psychopathology was the variable explaining the highest variation. Mediation analysis showed that negative symptoms mediate the effects of verbal fluency dysfunctions and high-level neurological soft signs (NSS) on TRS' disease severity. These results show that determinants of disease severity sharply differ in TRS and non-TRS patients, and let hypothesize that TRS may stem from cognitive disfunctions and putatively neurodevelopmental aberrations.

Evaluation of a few discrete clinical markers may predict categorization of actively symptomatic non-acute schizophrenia patients as treatment resistant or responders: A study by ROC curve analysis and multivariate analyses.

Here, we used Receiver Operating Characteristic (ROC) curve analysis to determine whether clinical factors may aid predicting the categorization of schizophrenia patients as Treatment Resistant (TRS) or antipsychotic responsive schizophrenia (ARS). Patients with an established condition of TRS or ARS were assessed for: clinical presentation and course; neurological soft signs (NES); psychopathology by PANSS; cognitive performances; quality of life scale (QLS); functional capacity; social functioning (PSP and SLOF scales). In ROC curve analysis, significance indicated that the Area under curve (AUC) allowed distinguishing between TRS and ARS. Multivariate analyses were additionally used to provide independent predictive analysis. Multiple clinical variables showed significant AUCs. The largest significant AUCs were found for: NES total score; SLOF Area2; QLS subscale; antipsychotic doses. The highest sensitivity was found for NES total score, the highest specificity for previous hospitalizations. The highest Odds Ratio of being included within the TRS category were found for: NES total score (7.5); QLS total score (5.49); and previous hospitalizations (4.76). This same circumscribed group of variables was also found to be predictive of TRS when adopting stepwise logistic regression or discriminant analysis. We concluded that the evaluation of few clinical factors may provide reliable and accurate predictions on whether one schizophrenia patient may be categorized as a TRS.

Treatment resistant schizophrenia and neurological soft signs may converge on the same pathology: Evidence from explanatory analysis on clinical, psychopathological, and cognitive variables.

Here, we investigated neurological soft signs (NSSs) in treatment resistant schizophrenia (TRS) vs treatment responder schizophrenia (SZ) patients. TRS is a severe condition, affecting approximately one-third of schizophrenia patients and representing a relevant clinical challenge. NSSs are neurological abnormalities reportedly described in schizophrenia patients and linked to dysregulated network connections. We explored the possibility that NSSs may be: i) more severe in TRS patients; ii) differentially associated to clinical/cognitive variables in TRS vs SZ; iii) predictive of having TRS. In addition, we evaluated whether diagnosis may mediate NSSs associations with the above-mentioned variables. Consecutive patients with schizophrenia diagnosis underwent stringent assessment for TRS diagnosis. Demographics and clinical variables were recorded. Psychopathology (by Positive and Negative Syndrome Scale, PANSS), cognitive performances, and NSSs (by Neurological Evaluation Scale, NES) were tested. TRS had higher scores than SZ patients in total NES score and in almost all NES subscales, even after correction for duration of illness and antipsychotic dose (ANCOVA, p<0.05). NSSs significantly correlated with multiple clinical, psychopathological, and cognitive variables (above all: duration of disease and negative symptoms) in TRS but not in SZ patients. Two-way ANOVA showed NSS-x-diagnosis interaction in determining outcomes on multiple cognitive performances, but not in other clinical variables. However, simple main effect analysis detected a significant relationship between high severity NSSs and TRS diagnosis on multiple clinical and cognitive outcomes. Hierarchical regression analysis showed that diagnosis was among a discrete number of predictors yielding significant increases in variance explained on NES total, Sensory Integration and Other Signs subscales' scores. NSSs, together with antipsychotic dose and disease severity, were found to be significantly predictive of TRS diagnosis in a binary logistic regression model. These results suggest a stringent association between NSSs and TRS diagnosis, and may imply that NSSs association with clinical, psychopathological, and cognitive variables may be in part mediated by TRS diagnosis.

Re-arrangements of gene transcripts at glutamatergic synapses after prolonged treatments with antipsychotics: A putative link with synaptic remodeling.

OBJECTIVES: The postsynaptic density (PSD) represents a site of dopamine-glutamate integration. Despite multiple evidence of PSD involvement in antipsychotic-induced synaptic changes, there are no direct head-to-head comparisons of the effects at the PSD of antipsychotics with different receptor profile and at different doses after chronic administration. METHODS: Molecular imaging of gene expression was used to investigate whether chronic treatment with first and second generation antipsychotics (haloperidol, asenapine and olanzapine) may induce changes in the expression levels of PSD transcripts involved in schizophrenia pathophysiology, i.e. Homers, Shank1, PSD-95 and Arc. RESULTS: Genes' expression patterns were differentially modulated after chronic administration of typical and atypical antipsychotics as well as by the same compound administered at different doses. Antipsychotic treatment reduced gene expression in cortical regions, while Homer1a was still induced in striatum by haloperidol even after prolonged treatment. Moreover, chronic treatments appeared to cause a "de-recruitment" of brain regions demonstrated to be activated in acute treatments, with a prominent effect in the cortex rather than in striatum. CONCLUSIONS: These results let hypothesize that prolonged antipsychotic treatment may trigger a set of plastic changes involving scaffolding and effector molecules causing a possible re-arrangement of PSD transcripts in brain regions relevant to schizophrenia pathophysiology.