Progress in Neuro-Psychopharmacology and Biological Psychiatry

Despite extensive epigenetic reprogramming, induced pluripotent stem cells (iPSC) from schizophrenia patients (SZ) retain several molecular and functional features of this disease. Transcriptomic and epigenomic analyses were performed in iPSC of SZ and healthy control subjects (HC). Transcriptional profiles were largely similar between SZ and HC iPSC, whereas pronounced differences emerged following neural differentiation, with SZ NSC exhibiting dysregulation of genes involved in neurodevelopment and synaptic function. ZNF5c0 was identified as a uniquely and consistently upregulated gene in SZ iPSC, robustly discriminating SZ from HC iPSC. Epigenomic profiling revealed increased chromatin accessibility and reduced DNA methylation at the ZNF5c0 promoter in SZ iPSC. ChIP-seq data suggested that ZNF560 can bind to promoters of genes implicated in synaptic signaling and neuronal development. Moreover, a subset of these genes was found to be differentially expressed in SZ neural stem cells. Together, our results identify ZNF5c0 as a reprogramming-resistant epigenetic marker of schizophrenia and suggest an altered KRAB-ZNF-mediated regulation in early neurodevelopmental pathways underlying this disorder.

The present study aims to describe the acute effects of 5-HT2C receptor agonists and antagonists in behavioral tests related to anxiety in adult zebrafish (Danio rerio). For this, three groups of fish (n = 12/group) were used: a group treated with the drug MK-212 (2 mg/kg), a 5-HT2C receptor agonist; another group treated with the drug RS-102221 (2 mg/kg), a 5-HT2C receptor antagonist; and a third group, the control group, using a vehicle solution. The three groups were exposed to two behavioral tests: novel tank test (NTT) and light-dark preference (LDT). MK-212 produced no effects on the NTT, while RS-102221 decreased geotaxis (d = 0.99, 95%CI[0.3, 1.7]) and erratic swimming (d = 0.87, 95%CI[0.18, 1.56]) in this test; these effects are consistent with a tonic facilitation of defensive behavior in the NTT. Conversely, in the LDT, MK-212 increased scototaxis (d = 0.96, 95%CI[0.25, 1.65]), risk assessment (d = -1.31, 95%CI[-2.02, -0.59]), and thigmotaxis (d = -1.48, 95%CI[-2.21, -0.75]), consistent with an anxiogenic-like effect of this drug. Thus, tonic and phasic effects of the activation of this receptor are observed depending on the type of behavioral test used.

Substantial research on the association between early-life stress and its long-lasting impact on lifetime mental health has been performed revealing that early-life environmental adversity strongly regulates brain function. Alterations of gene expression and behavior in the off-springs of paternally stressed rats were also revealed. However, the precise mechanisms underlying these changes remain poorly understood. Here, an improved characterization of these processes from investigations of the functional metabolism of animal models exposed to peripubertal stress (PS) is proposed. The ultimate goal of this study was to bring forward functional Magnetic Resonance Spectroscopy (fMRS) as a technique of interest for a better understanding of brain areas by endogenous stimulators such as stress. The present study evaluated, compared and classified effects of individual PS (iPS) and paternal PS (pPS) under corticosterone (CORT) challenge in the septal areas of adult rats. Acute stress was simulated by injection of CORT and metabolic concentration changes were analyzed as a function of time. Evaluation of Glucose and Lactate concentration changes allowed the classification of groups of rats using a Glc to Lac index. Moreover, metabolic responses of control rats (CC) and of pPS x iPS rats (SS) were similar while responses in pPS (SC) and iPS (CS) differed, revealing differential adaption of energetic metabolism and of glutamatergic neurotransmission. Findings have crucial interest for understanding the metabolic mechanisms underlying altered functional connectivity and neuronal plasticity in septal areas inducing increased aggressivity in early-life stressed rats.

Autism Spectrum Disorders (ASD) are characterized by core behavioral symptoms in the domains of sociability, language/communication, and repetitive or stereotyped behaviors. Deficits in the prefrontal and hippocampal excitatory/inhibitory balance due to a functional loss of GABAergic interneurons are proposed to underlie these symptoms. Increasing the postsynaptic effects of GABA with compounds that selectively modulate GABAergic receptors could be a potential target for treating ASD symptoms. In addition, deficits in GABAergic interneurons have been linked to dopamine (DA) system dysregulation, and, despite conflicting evidence, abnormalities in the DA system activity may underly some ASD symptoms. Here, we investigated whether the positive allosteric modulator of 5-containing GABAA receptors (5-GABAARs) SH-053-2F-R-CH3 (10 mg/kg) attenuates behavioral abnormalities in a rat model for autism based on in utero VPA exposure. We also evaluated if animals exposed to VPA in utero present changes in the ventral tegmental area (VTA) DA system activity using in vivo electrophysiology and if SH-053-2F-R-CH3 could attenuate these changes. In utero VPA exposure caused male and female rats to present increased repetitive behavior (self-grooming) in early adolescence and deficits in social interaction in adulthood. Male, but not female VPA rats, also presented deficits in recognition memory as adults. SH-053-2F-R-CH3 attenuated the impairments in sociability and cognitive function in male VPA-exposed rats without attenuating the decreased social interaction in females. Male and female adult VPA-exposed rats also showed an increased VTA DA neuron population activity, which was not changed by SH-053-2F-R-CH3. Despite sex differences, our findings indicate 5-GABAARs positive allosteric modulators may effectively attenuate some core ASD symptoms.

The social anxiety disorder (SAD) is one of the most common mental health disorders, often developing in adolescence. It can severely impair educational achievement, career progression, and social functioning. Certain individuals within the Chinese Kunming dog (CKD) population display spontaneous, SAD-like behaviors towards strangers, offering a valuable model for genetic investigation. Using whole-genome sequencing data, we conducted a genome-wide association study on this SAD-like behavior in 100 CKDs, identifying several genes previously linked to human psychiatric disorders. Notably, SLC6A3 reached genome-wide significance, whereas CADPS2 exceeded only the suggestive threshold and is therefore considered a network-supported candidate locus. By integrating weighted gene co-expression network analysis with public RNA-seq data from the VTA of 38 mice (after quality control), we further showed that Slc6a3 and Cadps2 are part of a co-expression network module associated with the dopamine system. These findings suggest that this specific module may play a functional role in the stranger-directed anxiety-related behaviours in CKDs. Furthermore, we identified Nrip3 as a key candidate transcriptional coregulator in this specific module. Subsequent virus-mediated overexpression and behavioural experiments showed that Nrip3 regulates gene expression within this network and induces a spectrum of behavioural alterations in mice, including social avoidance, anhedonia, and increased locomotor activity. Collectively, this study highlights the involvement of dopamine system genes in canine social anxiety and suggests Nrip3 as an upstream regulator of Slc6a3 associated with anxiety. The identification of conserved anxiety-related signatures between humans and dogs further supports the dog as a valuable translational model for psychiatric research.

Substance use disorders are more prevalent in schizophrenia, worsening its course and prognosis. Here, we used a double-hit rat model, combining maternal immune activation (MIA) and peripubertal stress (PUS), to study cocaine addiction and the underlying neurobehavioural alterations. We injected lipopolysaccharide or saline on gestational days 15 and 16 to pregnant rats. Their male offspring were then subjected to 5 episodes of unpredictable stress every other day during adolescence (from postnatal day 28 to 38). When rats reached adulthood, we studied cocaine addiction-like behaviour, impulsivity, conditioning processes and several aspects of brain structure and function by MRI, PET and RNAseq. MIA facilitated the acquisition of cocaine self-administration while PUS reduced cocaine intake, an effect that was reversed by MIA. MIA increased motivation for cocaine and reversed the effects of PUS during extended access. Incubation of seeking was unaffected. Neither hit alone nor their combination impacted Pavlovian or instrumental conditioning or impulsiveness. At the brain level, PUS reduced hippocampal volume and hyperactivated the dorsal subiculum. MIA+PUS altered the structure and function of the dorsal striatum increasing its volume and interfering with glutamatergic dynamics. MIA did not affect the gene expression of the nucleus accumbens but, when combined with PUS, modulated specific genes that could account for the restored cocaine intake. PUS had a profound effect on the dorsal striatal transcriptome however, this was obliterated when PUS occurred in animals with MIA. These results describe a complex interplay between MIA and stress on neurodevelopment and in the susceptibility to develop cocaine addiction.

Fragile X syndrome (FXS) is an X-linked developmental disorder characterized by several behavioral abnormalities, including hyperactivity, sensory hyper-responsiveness and cognitive deficits, as well as autistic symptoms, e.g., reduced social interaction. These behavioural alterations are recapitulated by the major animal model of FXS, i.e., the Fmr1-KO mouse, which has been extensively employed to identify therapeutic targets for FXS, though effective pharmacological treatments are still lacking. Here we focused on the therapeutic role of large-conductance Calcium-dependent potassium (BKCa) channels, playing a crucial role in neuronal excitability and neurotransmitter release. Reduced expression/functionality of these channels has been described in FXS patients and mice, so that molecules activating these channels have been proposed as promising treatments for this syndrome. Here we performed an extensive characterization of the therapeutic impact of a novel BKCa agonist on FXS-like symptoms in the Fmr1-KO mouse model, employing a drug repurposing setting. We evaluated the acute and chronic effects of chlorzoxazone, i.e., a classical drug used for non-developmental muscular pathologies, on the locomotor, social, cognitive and sensory-motor alterations of Fmr1-KO mice and compared them with other pharmacological treatments recently proposed for FXS that instead do not target BKCa channels. Our results clearly demonstrate for the first time the marked efficacy of chlorzoxazone in treating all the behavioral abnormalities of FXS mice, thus encouraging the preferential use of this molecule over others for clinical applications in the field of FXS, and potentially of other neurodevelopmental disorders.

The effects of previous social experiences on social behavior have been demonstrated across species both in cooperative and competitive contexts. In dominance-subordinate hierarchies, differences across social ranks have been observed in many different mechanisms. Dominance hierarchies interfere in defensive behavior, where subordinate animals present a greater defensive behavior, regarding potential threats ("anxiety-like behavior"), than dominant animals. The serotonergic system plays a key role in regulating and mediating threat responses, including 5-HT2 receptors in the types of proximal threat responses modulated by the stress of social defeat. We separated 148 adult zebrafish in pairs, and allowed to interact for five days; after that, the dominant-subordinate rank was determined, and animals were treated with a 5-HT2C receptor agonist (MK-212) or antagonist (RS-102221) before being observed in the novel tank test. While MK-212 increased bottom-dwelling, erratic swimming, and freezing across all statuses, RS-102221 decreased these variables in dominants but increased them in subordinates. Moreover, the effects of MK-212 were larger in subordinates than in controls or dominants, suggesting a sensitization of the 5-HT2C receptor.

Behavioral copying is a key process in group actions, but it is challenging for individuals with Autism Spectrum Disorder (ASD). We investigated behavioral contagion, or instinctual replication of behaviors, in Krushinky-Molodkina (KM) rats (n=16), a new rodent model for ASD, compared to control Wistar rats (n=15). A randomly chosen healthy Wistar male ("demonstrator rat") was introduced to the homecage of experimental rats ("observers") 10-14 days before the experiments to become a member of the group. For the implementation of the behavioral contagion experiment, we used the IntelliCage system, where rats can live in a group of 5-6 rats and their water visits can be fully controlled. During the experiment, the demonstrator was taken out of IntelliCage for 24 hours of water deprivation and then placed back. As a result, a drinking behavior of the water-deprived demonstrator rat prompted activated behaviors in the whole group. Unlike the Wistar controls, KM observers showed fewer visits to the drinking bottles, particularly lacking inspection visits. The control group, in contrast, exhibited a dynamic, cascade-like visiting of the water corners. The proportion of activated observers in KM rats was significantly lower, as compared to Wistar ones, and they did not mimic other observer rats. KM rats, therefore, displayed an attenuated pattern of behavioral contagion, highlighting social deficits in this ASD model. This study suggests that measuring group dynamics of behavioral contagion in an automated, non-invasive setup offers valuable insights into social behavior in rodents, particularly for studying social deficits in ASD models. HighlightsO_LIThirsty demonstrators triggered an avalanche of observers visits to the water corners C_LIO_LIThe contaged behavior was attenuated in observer KM rats C_LIO_LIBehavioral contagion test provides a new tool for objective, automated phenotyping in rodent models of social deficits C_LI

The Disrupted-in-schizophrenia 1 (DISC1) signaling pathway is considered to play a key role in schizophrenia, depression, autism and other psychiatric disorders. DISC1 is involved in regulating the dopaminergic neurotransmission in, among others, the mesolimbic reward system. A transgenic rat line tgDISC1 has been introduced as a model system to study behavioral phenotypes associated with abnormal DISC1 pathways. Here, we evaluated the impact of impaired DISC1 signaling on social (social interaction) and non-social (sucrose) reward preferences in the tgDISC1 animal model. In a plus-maze setting, rats chose between the opportunity for social interaction with an unfamiliar juvenile conspecific (social reward) or drinking sweet solutions with variable sucrose concentrations (non-social reward). tgDISC1 rats differed from wild-type rats in their social, but not in their non-social reward preferences. Specifically, DISC1 rats showed a lower interest in interaction with the juvenile conspecific, but did not differ from wild-type rats in their preference for higher sucrose concentrations. These results suggest that disruptions of the DISC1 pathway that is associated with altered dopamine transmission in the brain result in selective deficits in social motivation seen in neuropsychiatric illness.

This report is focusing on a function of the c-Fos protein in an associative, stress-induced memory. The shRNA vector injections were utilised to functionally silence the central amygdala nucleus in adult Wistar rats. Subsequently the operated animals and their control counterparts were screened in a selection of an emotionally-dependent tests and in a few standard behavioral neuroscience tools. Rats from the c-Fos silencing (ShFos) group expressed contra-depressive-like behaviors in Porsolt Swimming Test, spending more time actively searching for escape way then the rats from the control group. ShFos rats also had engaged in a more rapid activity in the Open Field Test, showing a decline in the neo-phobia. Micturition was decreased in shFos animals, indicating a change in the emotionality on an autonomic level. Presented results are showcasing a multi-directional regulation of the behaviors from the central amygdala nucleus by the c-Fos activity.

Nitric oxide (NO) is a molecule involved in plasticity across levels and systems. The role of NOergic pathways in time-dependent sensitization, a behavioral model of translational relevance to trauma and stress-related disorders, was assessed in adult zebrafish. In this model, adult zebrafish acutely exposed to a fear-inducing conspecific alarm substance (CAS) and left undisturbed for an incubation period show increased anxiety-like behavior 24 h after exposure. CAS increased forebrain glutamate immediately after stress and 30 min after stress, an effect that was accompanied by increased nitrite levels immediately after stress, 30 min after stress, 90 min after stress, and 24 h after stress. CAS also increased nitrite levels in the head kidney, where cortisol is produced in zebrafish. CAS-elicited nitrite responses in the forebrain 90 min (but not 30 min) after stress were prevented by a NOS-2 blocker. Blocking NOS-1 30 min after stress prevents TDS; blocking NOS-2 90 min after stress also prevents TDS, as does blocking calcium-activated potassium channels in this latter time window. TDS is also prevented by blocking guanylate cyclase activation in both time windows, and cGMP-dependent channel activation in the second time window. These results suggest that different NO-related pathways converge at different time windows of the incubation period to induce TDS.

Functional asymmetries in the medial prefrontal cortex (mPFC) are significant attributes of this brain area, implicated in its role in emotional processing and executive function. Evidence suggests that, under normal conditions, there is a tonic inhibition of the right (R) mPFC by the left (L) mPFC, and a dysregulation of this hemispheric functional lateralization is implicated in detrimental chronic stress effects. Considering the wide interhemispheric connection and the inhibitory tone from the LmPFC to the RmPFC, we hypothesize that alterations in the activity of the direct projections between the mPFC hemispheres during stressful situations are related to stress vulnerability. To address this question, we used a chemogenetic approach to modulate the activity of L-to-R dorsomedial prefrontal cortex (dmPFC) monosynaptic projections during psychosocial stress (PSS) exposure in mice. We found that activating LdmPFC projections during a repeated PSS protocol prevents stress-induced apathy-like behavior in females and males and social avoidance in male mice. On the other hand, inhibiting such projections during a single session of PSS increases vulnerability to stress effects in male mice, increasing social avoidance and anxiety-like behaviors. Both glutamatergic and GABAergic cells compose the projecting interhemispheric neurons in the dmPFC. However, the LdmPFC showed a higher density of glutamatergic projections to the RdmPFC than the opposite. In conclusion, our results revealed an involvement of the monosynaptic projections from the LdmPFC to RdmPFC in the vulnerability to the behavioral alterations induced by PSS in female and male mice.

Depressive disorders are one of the leading causes of non-fatal health loss in the last decade. Adding to the burden, the available treatments not always properly work for some individuals. There is, therefore, a constant effort from clinical and preclinical studies to bring forward a better understanding of the disease and look for novel alternative therapies. Two target systems very well explored are the serotonin and the brain-derived neurotrophic factor (BDNF) systems. Selective serotonin reuptake inhibitors (SSRIs), a commonly used class of antidepressants, target the serotonin transporter (SERT) and increase serotonin levels, which in turn also leads to an increase in BDNF. A rat model lacking SERT (SERT knockout) has been a useful tool to study the interplay between serotonin and BDNF. SERT-/- rats present increased extracellular levels of serotonin, yet BDNF levels are decreased, especially in the prefrontal cortex (PFC) and hippocampus. The animals further display anxiety- and depression-like behavior. Therefore, BDNF might mediate the phenotype expressed by the SERT-/- rats. In this study, we sought to investigate whether overexpression of BDNF in the brain of SERT-/- rats would rescue its anxious and depressive-like behavior. Through stereotaxic surgery, SERT-/- and wild-type (WT) rats received BDNF or GFP lentivirus microinfusions into the prelimbic cortex subregion of the mPFC and were submitted to the sucrose consumption, open field test, and forced swim tests. Additionally, we measured hypothalamus-pituitary-adrenal (HPA)-axis reactivity. The results revealed that SERT-/- rats presented decreased sucrose intake, decreased locomotor activity, and increased escape-oriented behavior in the forced swim test compared to WT rats. BDNF upregulation in WT rats caused alterations in the HPA-axis function, resulting in elevated basal plasma corticosterone levels and decreased plasma corticosterone upon stress. In conclusion, BDNF overexpression in the PrL, in general, did not rescue SERT-/- rats from its depression- and anxiety-like behavior, and in WT animals, it caused a malfunction in the HPA-axis.

Externalizing disorders (EDs) are characterized by outward-directed behaviors such as aggression and hyperactivity. They are influenced by gene-environment interactions, yet our understanding of the genetic predispositions and environmental contexts that give rise to them is incomplete. Additionally, people with EDs often exhibit comorbid internalizing symptoms, which can complicate the clinical presentation and treatment strategies. Following on from our previous studies, we examined genes x environment interaction as a risk factor for EDs by looking at internalizing and externalizing behaviors after social isolation. Specifically, we subjected adgrl3.1 knockout zebrafish -- characterized by hyperactivity and impulsivity -- to a 2-week social isolation protocol. We subsequently assessed the impact on anxiety-like behavior, abnormal repetitive behaviors, working memory, and social interactions. Genotype-specific additive effects emerged, with socially isolated adgrl3.1 knockout fish exhibiting intensified comorbid phenotypes, including increased anxiety, abnormal repetitive behaviors, reduced working memory, and altered shoaling, when compared to WT fish. The findings demonstrate that genetic predispositions interact with environmental stressors, such as social isolation, to exacerbate both externalizing and internalizing symptoms. This underlines the necessity for comprehensive diagnostic and intervention strategies.

BackgroundThe gamification of behavioral intervention for tic disorders (TDs) may not only enhance compliance with treatment protocols but also offer a key clinical advantage. By providing immediate positive feedback when tics are suppressed, games can counteract negative reinforcement processes that reinforce tics, which assumingly alleviates unpleasant premonitory urges. We developed a gamified protocol (XTics), which leverages this potential by combining gamified tic triggers with immediate reinforcement of tic suppression. We evaluate the clinical value of immediate reward contingency in enhancing tic suppression performance. MethodsXTics comprises two conditions: tic-contingent and non-contingent. In the ticcontingent version, game progression was determined by real-time input from an experimenter who monitored the participants tics, rewarding tic suppression with favorable outcomes. Conversely, in the non-contingent version, game events occurred randomly. Using a crossover design, we trained 35 participants (aged 7-15) in both versions, with each participant undergoing a preliminary behavioral training and three hourly sessions for each condition. We both evaluated the overall protocols four-week impact on tic severity measures and compared contingent and non-contingent conditions. ResultsWe achieved complete adherence to the protocol, while the participants increased their tic-free intervals by an average of 558% from the first to the last training day. YGTSS, a clinical measure of tic severity, showed an average clinically meaningful reduction of 25.69{+/-}23.39%, which was larger than that observed in control interventions and comparable to the effects of longer non-pharmacological treatments. Parent-reported tic severity decreased by 42.99{+/-}31.69% from baseline to three months post-treatment. When contrasting the ticcontingent with the tic-non-contingent training versions, we observed a larger improvement in tic-free interval duration in the former case (t(67)=3.15, p=.0025). Additionally, Rush, another measure of tic severity, demonstrated a greater reduction following training with the contingent compared to the non-contingent version (t(47)=3.47, p=.002). ConclusionThe combination of gamified tic triggering with immediate and contingent rewards demonstrates a promising approach for enhancing treatment efficacy in TDs, offering an engaging boost to traditional therapeutic methods.

BackgroundAlthough epigenetic dysregulation has long been proposed to promote the onset of schizophrenia, the landscape of the methylomic changes across the whole genome is yet established. MethodsUsing Infinium Human Methylation 850 BeadChip Array and MethylTarget sequencing method, we investigated the genome-wide methylation profiles and further validated methylation profiles of target genes in peripheral blood lymphocytes between individuals with psychosis risk syndrome (PRS), patients with first-episode schizophrenia (FES) and healthy controls (HC) in Chinese Han population. ResultsWe detected 372 sites between psychosis risk syndrome (PRS) and healthy controls (HC), which increased to 460 sites in first-episode schizophrenia (FES) with 207 sites shared. Both PRS and FES featured profound hypomethylation within gene body. Gene ontology and network annotation merged on loci enriched in disease associated signaling pathways (MAPK(Mitogen Activated Protein Kinases), Glutamatergic, GABAergic etc.). ConclusionsOur study implicated characteristic hypomethylation in both the discovery and validation cohorts in SYNGAP1, one of the frequently studied genes in neurodevelopmental disorders. This is the first methylome-wide association study between PRS and FES in Chinese Han population. Our findings provide potential biomarkers that can be used for future development of disease therapy and management.

Animal models are widely used to study stress-induced affective disorders and associated with them neuroinflammation and other neuroimmune processes. Here, we examined rat behavioral and hippocampal transcriptomic responses to prolonged chronic unpredictable stress (PCUS), as well as following a 4-week treatment with a classical antidepressant fluoxetine, an anti-inflammatory agent eicosapentaenoic acid (EPA), a pro-inflammatory agent lipopolysaccharide (LPS) and their combinations. Overall, PCUS evoked an anxiety-like behavioral phenotype in rats (corrected by chronic fluoxetine alone or combined with other drugs), EPA was anxiolytic and LPS promoted anxiety in this model. PCUS evoked pronounced transcriptomic responses in rat hippocampi, including >200 differentially expressed genes. While pharmacological manipulations did not affect hippocampal gene expression markedly, Gpr6, Drd2 and Adora2a were downregulated in stressed rats treated with fluoxetine+EPA, suggesting G protein-coupled receptor 6, dopamine D2 receptor and adenosine A2A receptor as potential evolutionarily conserved targets in chronic stress. Overall, these findings support the validity of rat PCUS paradigm as a useful tool to study stress-related affective pathologies and calls for further research probing how various conventional and novel drugs modulate behavioral and neurotranscriptomic biomarkers of chronic stress.

Depressive mothers often find the mother-child interaction to be challenging. Parental stress may further impair mother-child attachment, which may increase the risk of negative developmental consequences. We used rats with different vulnerability to depression (Wistar and Kyoto) to investigate the impact of stress (maternal separation-MS) on maternal behaviour and adolescent offspring cognition. MS in Kyoto dams increased pup-contact, resulting in higher oxytocin levels and lower anxiety-like behaviour after weaning, while worsening their adolescent offspring cognitive behaviour. Whereas MS in Wistar dams elicited higher quality of pup-directed behaviour, increasing Brain-Derived Neurotrophic Factor (BDNF) in the offspring, which seems to have prevented a negative impact on cognition. Hypothalamic oxytocin seems to impact the salience of the social environment cues (as negative for Kyoto) leading to different coping strategies. Our findings highlight the importance of contextual and individual factors in the understanding of the oxytocin role in modulating maternal behaviour and stress regulatory processes.

The chronic unpredictable mild stress (CUMS) paradigm is one of the most widely used preclinical models to investigate the role of stress in the neurobiology of depression and to evaluate potential antidepressant therapies, owing to its strong translational relevance. However, despite its extensive use, substantial discrepancies and concerns regarding the reproducibility of CUMS outcomes persist in the literature, with many studies reporting inconsistent or unreliable results. In the present comparative study, we implemented several CUMS protocols in mice that differed in the intensity and duration of stress exposure, and systematically evaluated the resulting pathophysiological and behavioural alterations associated with depressive- and anxiety-like phenotypes. We found that progressive escalation in stress intensity and frequency, as well as prolonged exposure, induced increasingly robust disease-related behavioural and physiological changes, including depression- and anxiety-like behaviours, adrenal hypertrophy, and reduced body weight gain.. Moreover, social isolation emerged as a major contributing factor exacerbating stress-induced deficits. Altogether, data suggest that a multidimensional assessment of the behavioural impairments is critical to reinforce the physiological changes indicative of a hyperactive hypothalamic-pituitary-adrenal axis (HPA). Differences in individual stress susceptibility, stressor intensity, frequency, and housing conditions are key determinants of CUMS outcomes. Therefore, precise, detailed, and transparent reporting of protocol parameters is critical to enhance reproducibility and enable meaningful comparisons across studies.