Psychotropic medication prescription rates, particularly of the selective serotonin reuptake inhibitor fluoxetine (FLX; Prozac), are on the rise in the female adolescent population. Particularly, because FLX is dispensed for the treatment of numerous mood-related illnesses, premenstrual dysphoric disorder, as well as for pain management. Although FLX is deemed efficacious shortly post/during chronic treatment, the potential for unexpected long-term side effects has not been thoroughly assessed. For this reason, in this brief report, we examined whether exposure to FLX, during adolescence, influences thermal nociception in adulthood. To do this, postnatal day (PD)-35 female C57BL/6 mice were exposed to FLX (250 mg/L in drinking water) for 15 consecutive days (PD35-49). Once mice reached adulthood (PD70) they were evaluated on thermal nociception sensitivity adopting the hot plate test. We found that adult mice with FLX history displayed reductions in body weight (g) as well as reduced time (s) to display hindpaw-licking behavior, when compared to controls. These outcomes suggest that juvenile FLX exposure induces thermal hyperalgesia in adulthood, thus questioning the safety of antidepressant exposure during early-life stages of development in the female population.

Depression denotes a dysregulated stress response with significant mental and health implications. This review examines the neurobiological mechanisms underlying depression and resilience, focusing on how stress mediators influence vulnerability to severe stressors contrasted with resilience. We analyze structural and functional alterations in key brain regions, genetic factors, and potential therapeutic interventions. Understanding these mechanisms offers insights into preventing depression onset instead of solely treating its manifestations.

Major Depressive Disorder (MDD) is a devastating, multifactorial disease with limited pharmacological treatment options. Patients with MDD exhibit alterations in their dopamine (DA) signaling pathways through the midbrain ventral tegmental area (VTA). A similar observation is also detected in preclinical models of stress - mice exhibit behavioral and physiological impairments following chronic social defeat stress (CSDS). Prior studies demonstrate that CSDS-susceptible mice have increased VTA DA neuronal excitability, in part driven by an upregulation in hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Inhibiting HCN channels with known inhibitors such as Cilobradine alleviates the negative behavioral effects of CSDS. Here, we aimed to identify Cilobradine analogs with improved neural tropism and inhibitory efficacy. Two compounds, MS7710 and MS7712, differing by their left-hand side moieties, have a similar, potent inhibitory effect on VTA DA Ih currents as compared to Cilobradine, and a greater inhibitory effect than Cilobradine on VTA DA firing rate. We demonstrate that MS7710 and MS7712 have superior brain/plasma concentration ratios as compared to Cilobradine. They were efficacious at inhibiting VTA DA neuron firing rate and bursting activity in CSDS-susceptible male mice at lower doses than Cilobradine, which was recapitulated in female CSDS-susceptible mice with MS7710. Finally, we define that a single intraperitoneal injection of MS7710 ameliorates CSDS-induced social interaction deficits and reward-associated cognitive inflexibility for at least two weeks in male and female mice. These findings yield a novel HCN channel inhibitor with improved neural tropism and stress-alleviating effects that could provide a basis for future antidepressant drug discovery.

Psychosocial stress can lead to emotional disorders and memory-related cognitive impairments. Evidence suggests that stress effects vary with age and sex, involving brain structures such as the medial prefrontal cortex (mPFC), amygdala, and hippocampus. This study hypothesized that witnessing social defeat stress (WSDS) during adolescence or adulthood would produce anxiety- and depression-like behaviors and cognitive deficits in adulthood, with outcomes affected by sex. We examined WSDS effects on male and female mice exposed during adolescence or adulthood, assessing: (i) social avoidance in the social interaction test, (ii) anxiety in the elevated plus-maze (EPM) and open field tests, (iii) cognition in the object recognition test, (iv) depression-like behaviors in the sucrose splash test, and (v) ΔFosB expression in neurons within the mPFC, basolateral amygdala (BLA) and dorsal hippocampus (DH). WSDS during adolescence resulted in reduced EPM open-arm exploration in both sexes and impaired novel object discrimination in males. In adulthood, WSDS reduced open-arm entries only in females and impaired novel object discrimination in both sexes. Female mice showed higher mPFC ΔFosB labeling than males, while control males exhibited higher labeling in the BLA and DH, which was not observed in WSDS mice. In conclusion, this study shows that WSDS during adolescence or adulthood induces anxiety-like behavior in both sexes, cognitive impairments in males, and sex-specific patterns of neuronal activation.

A substantial number of patients exhibit treatment-resistant depression (TRD), posing significant challenges to clinicians. The discovery of novel molecules or mechanisms that may underlie TRD pathogenesis and antidepressant actions is highly needed.

Using the PubMed database, the authors searched for emerging evidence of novel approaches for TRD based on experimental and human studies. Herein, the authors discuss the mechanisms underlying glutamatergic antagonists, modulators of the opioid system, and tryptamine-derivate psychedelics as well as the emerging platforms to investigate novel pharmacological targets for TRD. A search for clinical trials investigating novel agents and interventions for TRD was also conducted.

The understanding of the multiple pathophysiological mechanisms involved in TRD may add further value to the effective treatment, contributing to a more personalized approach. Esketamine was approved for the treatment of TRD and novel drugs with rapid antidepressant actions such as psilocybin and buprenorphine have also been investigated as potential therapeutic strategies. Over the past decades, technological advances such as omics approaches have broadened our knowledge regarding molecular and genetic underpinnings of complex conditions like TRD. Omics approaches could open new avenues for investigating glial-mediated mechanisms, including their crosstalk with neurons, as therapeutic targets in TRD.

The medial amygdala (MeA) is activated by social stimuli and manipulations of the MeA disrupt a wide range of social behaviors. Social stress can shift social behaviors and may accomplish this partly via effects on the MeA. However, very little is known about the effects of social stress on the electrophysiological activity of MeA neurons. The posterior division of the MeA (MeAp) has been implicated in driving social engagement. We hypothesized that repeated social stress would cause parallel changes in in vivo activity of MeAp neurons and social behavior. The resident-intruder paradigm was used to produce repeated social stress in adult male rats. After repeated social stress, MeAp neurons were recorded with in vivo single-unit electrophysiology in anesthetized rats. MeAp neurons, specifically those in the posterodorsal subnucleus (MeApd), fired faster in stressed rats than in controls, and this effect was directly associated with stressor intensity. The MeAp sends dense projections to the posterior bed nucleus of stria terminalis (pBNST) and ventromedial hypothalamus (VMH), and both regions are essential for social engagement and are sensitive to social stressors. MeAp projections to pBNST had higher activity after stress, whereas projections to the VMH were not affected. These effects were significant only in rats that displayed susceptibility to this social stressor, as demonstrated by lower weight gain. Furthermore, the effect of stress on MeApd and MeAp-pBNST neuronal firing was correlated with lower social interaction. These results indicate that heightened MeApd and MeA-pBNST activity may contribute to alterations in social behaviors following social stress.NEW & NOTEWORTHY Social stress contributes to psychiatric disorders and impacts multiple brain regions. However, effects on a crucial area for social function, the medial amygdala (MeA), are unclear. We found that social stress increased firing of posterior MeA neurons, and particularly neurons that project to bed nucleus of the stria terminalis, a region implicated in anxiety. Effects of stress on this circuit were associated with diminished social interaction and help clarify how stress can impact social functions.

Adjustment disorder (AD) is common among medically ill patients, yet current evaluation methods do not address the specific characteristics in this population. This study aimed to develop a measurement scale for AD in medically ill patients in Colombia and to find evidence of its validity and reliability.

This was a scale development and validation study. In the first qualitative phase, items were developed. In the second phase, the content validity of each item was evaluated by patients and clinicians. In the third phase, structural validity, internal consistency, test-retest reliability, criterion validity, and convergent construct validity were assessed. Items were analyzed using a generalized partial credit model within an item response theory framework.

The Adjustment Disorder Scale for Medically Ill Patients (ETAM, for its acronym in Spanish) was developed, comprising 20 items that address the free description of stressful situations in the last 15 days and mental symptoms attributed to them. Evidence of content validity was found. The scale was administered to 512 medically ill patients, revealing a three-dimensional structure: 1) "AD Symptoms", 2) "Impact on Self-Care", and 3) "Impact on Desire to Live". Internal consistency was adequate, with McDonald's omega of 0.95 and Cronbach's alpha between 0.82 and 0.92 for its dimensions. ETAM had high test-retest reliability (intraclass correlation coefficient of 0.98). Criterion validity evidence was obtained with an independent psychiatrist's diagnosis, with an AUROC of 0.99, and convergent validity was consistent with hypotheses of correlation with other instruments with similar constructs. Discrimination and difficulty parameters were calculated for each item.

The ETAM is a scale with evidence of validity and reliability that can be used for the diagnosis of AD in medically ill patients in Colombia.

Depression is presented as a multi-factorial bio-psycho-social expression that has evolved primarily as an effect of stressors related to the motivational/emotional systems that regulate the BrainMind in our relationship with conspecifics. These stressors may be caused by two sources of threat, firstly, the loss of bonding with the caregiver and later with a partner and/or group which relates to the SEPARATION (PANIC/GRIEF) system, secondly, social defeat as an expression of the social competition and social dominance. The sexual maturity drives the individual to social competition and social dominance, even if the latter often occurs before sexual maturity, e.g., chickens, dogs, non-human primates, and humans. Depression is an evolutionarily conserved mechanism in mammals to terminate both separation anxiety, so as to protect the vulnerable social brain from the consequences of prolonged separation anxiety, and the stress of social competition when social defeat is predictable. Adolescence and Young adulthood are particularly susceptible to these two types of threat because of human developmental characteristics that are summarized by the term neoteny. This refers to the slowing down of growth and development, resulting in both a prolonged period of dependence on a caring/protective adult and the persistence of juvenile characteristics throughout life. Therefore, neoteny makes the transition from childhood to sexual maturity more dramatic, making the integration of the SEPARATION (PANIC/GRIEF) system with the dynamics of social competition and dominance more stressful and a source of depression. Stress is an expression of the HPA-Hypothalamic-Pituitary-Adrenal axis that articulates with other systems, mainly the autonomic nervous system and the immune-inflammatory system. The latter is believed to be one of the most significant components in the dynamics of depressive processes, connected to the prodromes of its activation in childhood, under the pressure of environmental and relational stressors which can lead to learned helplessness. The recurrence of stressors makes it easier for the immune-inflammatory system to be activated in later life, which could make a significant contribution to the establishment of a depressive disease. The possible contribution of children's identification processes with their parents' depressive personalities through observational learning is considered.

Previously, a chronic social defeat stress (CSDS) model has been widely-adopted for assessing depressive-like behaviors in animals. However, there is still room for improvement in the CSDS model to safeguard study accuracy and the welfare of lab rodents. Our study team developed a novel, standardized apparatus to induce CSDS in rodents and assessed the model's practical adaptability.

An innovative CSDS cage apparatus and water bottle was designed. To evaluate the effectiveness of the newly developed tools, a variety of animal models, including the tail suspension test (TST), sucrose preference test, forced swimming test (FST), novelty-suppressed feeding test, female urine sniffing test, and open field test (OFT), were adopted to assess depressive-like behaviors in mice. Fluoxetine treatment was also administered to observe the reversal effect, as part of the validation.

The CSDS cage apparatus resulted in the manifestation of depressive-like behaviors in the model mice. Significant reductions in sucrose preference and urine sniffing time were observed, while the OFT revealed decreased central zone total distance, residence time, and frequency of entry. Moreover, increased immobility was found in the FST and TST. Fluoxetine treatment was found to successfully reverse the modeling effect.

The CSDS cage apparatus was validated for enhanced usability and addressed the previous challenges of water bottle leakage and lab rodent welfare issues. The consistent results from multiple behavioral tests also supported real-world application of the apparatus, offering researchers a promising alternative to conventional rodent cages.

There is a growing body of evidence implicating gut-brain axis dysfunction in the pathophysiology of mood disorders. Accordingly, gut microbiota has become a promising target for the development of biomarkers and novel therapeutics for bipolar and depressive disorders.

We describe the observed changes in the gut microbiota of patients with mood disorders and discuss the available studies assessing microbiota-based strategies for their treatment.

Microbiota-targeted interventions, such as symbiotics, prebiotics, paraprobiotics, and fecal microbiota transplants seem to attenuate the severity of depressive symptoms. The available results must be seen as preliminary and need to be replicated and/or confirmed in larger and independent studies, also considering the pathophysiological and clinical heterogeneity of mood disorders.

Chronic social defeat stress is a widely used depression model in male mice. Several proposed adaptations extend this model to females with variable, often marginal effects. We examined if the widely used male-defined metrics of stress are suboptimal in females witnessing defeat.

Using a data-driven method, we comprehensively classified social interaction behavior in 761 male and female mice after chronic social witness/defeat stress, examining social modulation of behavior and associations with conventional metrics (i.e., social interaction ratio).

Social stress induced distinct behavioral adaptation patterns in defeated males and witness females. Social interaction ratio led to underpowered analyses in witness females with limited utility to differentiate susceptibility/resilience. Data-driven analyses revealed changes in social adaptation in witness females that were captured in attenuated velocity change from no target to target trials. We explored the utility of this metric in 4 female social stress models and in male witnesses. Combining social interaction ratio and velocity change optimally differentiated susceptibility/resilience in witness females and revealed resilient-specific adaptation in a resilience-associated neural circuit in female mice.

Chronic witness stress induced behavioral changes in females that were qualitatively distinct from those observed in defeated males and not adequately sampled by standard male-defined metrics. Modulation of locomotion is a robust and easily implementable metric for rigorous research in witness female mice. Overall, our findings highlight the need to critically evaluate sex differences in behavior and implement sex-based considerations in preclinical model design.

Psychological stress during early development and adolescence may increase the risk of psychiatric and cardiometabolic comorbidities in adulthood. The gut microbiota has been associated with mental health problems such as depression and anxiety and with cardiometabolic disease, but the potential role of the gut microbiota in their comorbidity is not well understood. We investigated the effects and mode of action of the intestinal bacterium Christensenella minuta DSM 32891 on stress-induced mental health and cardiometabolic disturbances in a mouse model of social defeat stress. We demonstrate that administered C. minuta alleviates chronic stress-induced depressive, anxiogenic and antisocial behavior. These effects are attributed to the bacterium's ability to modulate the hypothalamic-pituitary-adrenal axis, which mediates the stress response. This included the oversecretion of corticosterone and the overexpression of its receptors, as well as the metabolism of dopamine (DA) and the expression of its receptors (D1, D2L and D2S). Additionally, C. minuta administration reduced chronically induced inflammation in plasma, spleen and some brain areas, which likely contribute to the recovery of physical and behavioral function. Furthermore, C. minuta administration prevented chronic stress-induced cardiovascular damage by regulating key enzymes mediating liver fibrosis and oxidative stress. Finally, C. minuta increased the abundance of bacteria associated with mental health. Overall, our study highlights the potential of microbiota-directed interventions to alleviate both the physical and mental effects of chronic stress.

Chronic stress has a profound impact on circadian regulation of physiology. In turn, disruption of circadian rhythms increases the risk of developing both psychiatric and metabolic disorders. To explore the role of chronic stress in modulating the links between neural and metabolic rhythms, we characterized the circadian transcriptional regulation across different brain regions and the liver as well as serum metabolomics in mice exposed to chronic social defeat stress, a validated model for studying depressive-like behaviors.

Male C57BL/6J mice underwent chronic social defeat stress, and subsequent social interaction screening identified distinct behavioral phenotypes associated with stress resilience and susceptibility. Stressed mice and their control littermates were sacrificed every 4 hours over the circadian cycle for comprehensive analyses of the circadian transcriptome in the hypothalamus, hippocampus, prefrontal cortex, and liver together with assessments of the circadian circulatory metabolome.

Our data demonstrate that stress adaptation was characterized by reprogramming of the brain as well as the hepatic circadian transcriptome. Stress resiliency was associated with an increase in cyclic transcription in the hypothalamus, hippocampus, and liver. Furthermore, cross-tissue analyses revealed that resilient mice had enhanced transcriptional coordination of circadian pathways between the brain and liver. Conversely, susceptibility to social stress resulted in a loss of cross-tissue coordination. Circadian serum metabolomic profiles corroborated the transcriptome data, highlighting that stress-resilient mice gained circadian rhythmicity of circulating metabolites, including bile acids and sphingomyelins.

This study reveals that resilience to stress is characterized by enhanced metabolic rhythms and circadian brain-liver transcriptional coordination.

There is a gap in existing knowledge of stress-triggered neurochemical and behavioral adaptations in females. This study was designed to explore the short-term consequences of a single social defeat (SD) on accumbal dopamine (DA) dynamics and related behaviors in female Wistar rats. During the SD procedure, rats demonstrated different stress-handling strategies, which were defined as active and passive coping. The "active" subjects expressed a significantly higher level of activity directed toward handling stress experience, while the "passive" ones showed an escalated freezing pattern. Remarkably, these opposite behavioral manifestations were negatively correlated. Twenty-four hours following the SD exposure, decreased immobility latency in the Porsolt test and cognitive augmentation in the new object recognition evaluation were evident, along with an increase in electrically evoked mesolimbic DA release in passive coping rats. Rats exhibiting an active pattern of responses showed insignificant changes in immobility and cognitive performance as well as in evoked mesolimbic DA response. Furthermore, the dynamics of the decline and recovery of DA efflux under the depletion protocol were significantly altered in the passive but not active female rats. Taken together, these data suggest that female rats with a passive coping strategy are more susceptible to developing behavioral and neurochemical alterations within 24 h after stress exposure. This observation may represent both maladaptive and protective responses of an organism on a short timescale.

The lateral habenula (LHb) has emerged as a pivotal brain region implicated in depression, displaying hyperactivity in human and animal models of depression. While the role of LHb efferents in depressive disorders has been acknowledged, the specific synaptic alterations remain elusive. Here, employing optogenetics, retrograde tracing, and ex vivo whole-cell patch-clamp techniques, we investigated synaptic transmission in male mice subjected to chronic social defeat stress (CSDS) at three major LHb neuronal outputs: the dorsal raphe nucleus (DRN), the ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTg). Our findings uncovered distinct synaptic adaptations in LHb efferent circuits in response to CSDS. Specifically, CSDS induced in susceptible mice postsynaptic potentiation and postsynaptic depression at the DRN and VTA neurons, respectively, receiving excitatory inputs from the LHb, while CSDS altered presynaptic transmission at the LHb terminals in RMTg in both susceptible and resilient mice. Moreover, whole-cell recordings at projection-defined LHb neurons indicate decreased spontaneous activity in VTA-projecting LHb neurons, accompanied by an imbalance in excitatory-inhibitory inputs at the RMTg-projecting LHb neurons. Collectively, these novel findings underscore the circuit-specific alterations in LHb efferents following chronic social stress, shedding light on potential synaptic adaptations underlying stress-induced depressive-like states.

Social recognition is an essential part of social function and often promotes specific social behaviors based on prior experience. Social and defensive behaviors in particular often emerge with prior experiences of familiarity or novelty/stress, respectively. This is also commonly seen in rodents toward same-strain and interstrain conspecifics. Medial amygdala (MeA) activity guides social choice based on age and sex recognition and is sensitive to social experiences. However, little is known about whether the MeA exhibits differential responses based on strain or how this is impacted by experience. Social stress impacts posterior MeA (MeAp) function and can shift measures of social engagement. However, it is unclear how stress impacts MeAp activity and contributes to altered social behavior. The primary goal of this study in adult male Sprague Dawley rats was to determine whether prior stress experience with a different-strain (Long-Evans) rat impacts MeAp responses to same-strain and different-strain conspecifics in parallel with a change in behavior using in vivo fiber photometry. We found that MeAp activity was uniformly activated during social contact with a novel same-strain rat during a three-chamber social preference test following control handling but became biased toward a novel different-strain rat following social stress. Socially stressed rats also showed initially heightened social interaction with novel same-strain rats but showed social avoidance and fragmented social behavior with novel different-strain rats relative to controls. These results indicate that heightened MeAp activity may guide social responses to novel, threatening, rather than non-threatening, social stimuli after stress.

The rostral ventromedial medulla (RVM) is a crucial structure in the descending pain modulatory system, playing a key role as a relay for both the facilitation and inhibition of pain. The chronic social defeat stress (CSDS) model has been widely used to study stress-induced behavioral impairments associated with depression in rodents. Several studies suggest that CSDS also causes changes related to chronic pain. In this study, we aimed to investigate the involvement of the RVM in CSDS-induced behavioral impairments, including those associated with chronic pain. We used chemogenetics to activate or inhibit the RVM during stress. The results indicated that the RVM is a vital hub influencing stress outcomes. Rostral ventromedial medulla activation during CSDS ameliorates all the stress outcomes, including social avoidance, allodynia, hyperalgesia, anhedonia, and behavioral despair. In addition, RVM inhibition in animals exposed to a subthreshold social defeat stress protocol induces a susceptible phenotype, facilitating all stress outcomes. Finally, chronic RVM inhibition-without any social stress stimulus-induces chronic pain but not depressive-like behaviors. Our findings provide insights into the comorbidity between chronic pain and depression by indicating the involvement of the RVM in establishing social stress-induced behavioral responses associated with both chronic pain and depression.

Studies in adults have linked stress-related activation of the immune system to the manifestation of psychiatric conditions. Using a translational design, this study aimed to examine the impact of social stress on immune activity in adolescents and on neuronal activity in a preclinical mouse model. Participants were 31 adolescents (ages 12-19), including 25 with mood and anxiety symptoms. Whole-blood samples were collected before and after the Trier Social Stress Test (TSST), a stress-inducing public speaking task, then cultured for 6 hours in the presence and absence of the inflammatory endotoxin lipopolysaccharide (LPS). Effects of TSST and LPS on 41 immune biomarkers were examined using repeated-measures analysis of variance. Separately, juvenile (8-week-old) male mice were non-stressed or exposed to reminder social defeat then intraperitoneally injected with saline or LPS (n = 6/group). Brains were perfused and collected for immunohistochemistry and confocal microscopy at 0, 1, 6, and 24 hours post-injection. The activity was determined by the density of cFos-positive neurons in the paraventricular hypothalamus, paraventricular thalamus, and basolateral amygdala, regions known to show sustained activation to immunological challenge. Analyses in the adolescent study indicated a strong effect of LPS but no effects of TSST or TSST×LPS interaction on immune biomarkers. Similarly, reminder social defeat did not induce sustained neuronal activity changes comparable to LPS immunological challenge in juvenile mice. Our convergent findings across species suggest that the acute immune response to stress documented in adults is not present in youth. Thus, aging and chronicity effects may play an important role in the inflammatory response to acute psychosocial stress.

The social defeat paradigm is the most representative animal model to study social anxiety disorder (SAD) and its underlying neuronal mechanisms. We have previously reported that defeat progressively reduces oxytocin receptors (OXTR) in limbic regions of the brain over an eight-week period in female prairie voles (Microtus ochrogaster). Oxytocin receptors activate the mitogen-activated protein kinase (MAPK) pathway, which has been previously associated with the anxiolytic effects of oxytocin. Here, we assessed the functional significance of OXTR in stress-induced social avoidance and the response of the MAPK signaling pathway in the nucleus accumbens (NAc), anterior cingulate cortex (ACC), and basolateral amygdala (BLA) of female prairie voles.

In experiment 1, Sexually naïve adult female prairie voles were defeated for three consecutive days and tested a week after for social preference/avoidance (SPA) test. Control subjects were similarly handled without defeat conditioning. In experiment 2, sexually and stress naïve adult female prairie voles were bilaterally injected into the NAc, ACC, or the BLA with a CRISPR/Cas9 virus targeting the Oxtr coding sequence to induce OXTR knockdown. Two weeks post-surgery, subjects were tested for SPA behavior. Viral control groups were similarly handled but injected with a control virus. A subgroup of animals from each condition in both experiments were similarly treated and euthanized without being tested for SPA behavior. Brains were harvested for OXTR autoradiography, western blot analysis of MAPK proteins and quantification of local oxytocin content in the NAc, BLA, ACC, and PVN through ELISA.

Social defeat reduced OXTR binding in the NAc and affected MAPK pathway activity and oxytocin availability. These results were region-specific and sensitive to exposure to the SPA test. Additionally, OXTR knockdown in the NAc, ACC, and BLA induced social avoidance and decreased basal MAPK activity in the NAc. Finally, we found that OXTR knockdown in these regions was associated with less availability of oxytocin in the PVN.

Dysregulation of the oxytocin system and MAPK signaling pathway in the NAc, ACC, and BLA are important in social behavior disruptions in female voles. This dysregulation could, therefore, play an important role in the etiology of SAD in women.

Depression is a prevalent psychiatric disorder with accumulating evidence implicating dysregulation of extracellular adenosine triphosphate (ATP) levels in the medial prefrontal cortex (mPFC). It remains unclear whether facilitating endogenous ATP production and subsequently increasing extracellular ATP level in the mPFC can exert a prophylactic effect against chronic social defeat stress (CSDS)-induced depressive-like behaviors and enhance stress resilience. Here, we found that nicotinamide mononucleotide (NMN) treatment effectively elevated nicotinamide adenine dinucleotide (NAD+) biosynthesis and extracellular ATP levels in the mPFC. Moreover, both the 2-week intraperitoneal (i.p.) injection and 3-week oral gavage of NMN prior to exposure to CSDS effectively prevented the development of depressive-like behavior in mice. These protective effects were accompanied with the preservation of both NAD+ biosynthesis and extracellular ATP level in the mPFC. Furthermore, catalyzing ATP hydrolysis by mPFC injection of the ATPase apyrase negated the prophylactic effects of NMN on CSDS-induced depressive-like behaviors. Prophylactic NMN treatment also prevented the reduction in GABAergic inhibition and the increase in excitability in mPFC neurons projecting to the lateral habenula (LHb). Collectively, these findings demonstrate that the prophylactic effects of NMN on depressive-like behaviors are mediated by preventing extracellular ATP loss in the mPFC, which highlights the potential of NMN supplementation as a novel approach for protecting and preventing stress-induced depression in susceptible individuals.

Psychedelic compounds have potentially rapid, long-lasting anxiolytic, antidepressive and anti-inflammatory effects. We investigated whether the psychedelic compound (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI], a selective 5-HT2A receptor partial agonist, decreases stress-related behavior in male mice exposed to repeated social aggression. Additionally, we explored the likelihood that these behavioral changes are related to anti-inflammatory properties of [(R)-DOI]. Animals were subjected to the Stress Alternatives Model (SAM), an escapable social stress paradigm in which animals develop reactive coping strategies - remaining in the SAM arena (Stay) with a social aggressor, or dynamically initiated stress coping strategies that involve utilizing the escape holes (Escape) to avoid aggression. Mice expressing these behavioral phenotypes display behaviors like those in other social aggression models that separate animals into stress-vulnerable (as for Stay) or stress-resilient (as for Escape) groups, which have been shown to have distinct inflammatory responses to social stress. These results show that Stay animals have heightened cytokine gene expression, and both Stay and Escape mice exhibit plasma and neural concentrations of the inflammatory cytokine tumor necrosis factor-α (TNFα) compared to unstressed control mice. Additionally, these results suggest that a single administration of (R)-DOI to Stay animals in low doses, can increase stress coping strategies such as increasing attention to the escape route, promoting escape behavior, and reducing freezing during socially aggressive interaction in the SAM. Lower single doses of (R)-DOI, in addition to shifting behavior to suggest anxiolytic effects, also concomitantly reduce plasma and limbic brain levels of the inflammatory cytokine TNFα.

The Hypothalmic-Pituitary-Adrenal axis also known as the HPA axis is central to stress response. It also acts as the relay center between the body and the brain. We analysed hypothalamic proteome from mice subjected to chronic social defeat paradigm using iTRAQ based quantitative proteomics to identify changes associated with stress response. We identified greater than 2000 proteins after processing our samples analysed through Q-Exactive (Thermo) and Orbitrap Velos (Thermo) at 5% FDR. Analysis of data procured from the runs showed that the proteins whose levels were affected belonged primarily to mitochondrial and metabolic processes, translation, complement pathway among others. We also found increased levels of fibrinogen, myelin basic protein (MBP) and neurofilaments (NEFL, NEFM, NEFH) in the hypothalamus from socially defeated mice. Interestingly, research indicates that these proteins are upregulated in blood and CSF of subjects exposed to trauma and stress. Since hypothalamus secreted proteins can be found in blood and CSF, their utility as biomarkers in depression holds an impressive probability and should be validated in clinical samples.

Inflammasome complexes, especially NLRP3, have gained great attention as a potential therapeutic target in mood disorders. NLRP3 triggers a caspase 1-dependent release of the inflammatory cytokines IL-1β and IL-18, and seems to interact with purinergic and kynurenine pathways, all of which are implicated in mood disorders development and progression.

Emerging evidence supports NLRP3 inflammasome as a promising pharmacological target for mood disorders. We discussed the available evidence from animal models and human studies and provided a reflection on drawbacks and perspectives for this novel target.

Several studies have supported the involvement of NLRP3 inflammasome in MDD. However, most of the evidence comes from animal models. The role of NLRP3 inflammasome in BD as well as its anti-manic properties is not very clear and requires further exploration. There is evidence of anti-manic effects of P2×R7 antagonists associated with reduction in the brain levels of IL-1β and TNF-α in a murine model of mania. The involvement of other NLRP3 inflammasome expressing cells besides microglia, like astrocytes, and of other inflammasome complexes in mood disorders also deserves further investigation. Preclinical and clinical characterization of NLRP3 and other inflammasomes in mood disorders is needed before considering translational approaches, including clinical trials.

Pharmacological inhibition of fatty acid amide hydrolase (FAAH) activity has antidepressant-like effects in preclinical models of stress. In this study, we investigated whether the antidepressant-like effects of FAAH inhibition are associated with corresponding changes in gut microbial and lipidomic profiles, which are emerging as critical components in the pathophysiology of depression. Adult male Wistar rats experienced five weeks of repeated social defeat or control procedure and were treated with the FAAH inhibitor URB694 (0.3 mg/kg/day, i.p.) or vehicle starting from the third week. Repeated social defeat induced the emergence of depressive-like behavioral (sucrose preference reduction and passive coping behaviors in the forced swim test) and neuroendocrine (increased corticosterone levels) changes, which were prevented by URB694 treatment. Repeated social defeat also provoked a significant variation in gut microbiota (changes in the relative abundance of 14 bacterial taxa) and lipidic (e.g., glycerophospholipids) composition. These stress-induced changes were prevented by URB694 treatment. These findings indicate that inhibition of FAAH activity with URB694 blocks the co-occurrence of depressive-like behavioral and neuroendocrine changes and alterations in gut microbial and lipid composition in rats exposed to repeated social defeat. In conclusion, these results suggest that the gut microbiota-lipid crosstalk may represent a novel biological target for FAAH inhibitors to enhance stress resilience.

Stress-induced illnesses, like major depression, are among the leading causes of disability across the world. Consequently, there is a dire need for the validation of translationally-suited animal models incorporating social stress to uncover the etiology of depression. Prairie voles (Microtus ochrogaster) are more translationally relevant than many other rodent models as they display monogamous social and bi-parental behaviors. Therefore, we evaluated whether a novel social defeat stress (SDS) model in male prairie voles induces depression-relevant behavioral outcomes.

Adult sexually-naïve male prairie voles experienced SDS bouts from a conspecific pair-bonded male aggressor, 10 min per day for 10 consecutive days. Non-stressed controls (same-sex siblings) were housed in similar conditions but never experienced physical stress. Twenty-four h later, voles were evaluated in social interaction, sucrose preference, and Morris water maze tests - behavioral endpoints validated to assess social withdrawal, anhedonia-related behavior, and spatial memory performance, respectively.

SDS-exposed voles displayed lower sociability and body weight, decreased preference for a sucrose solution, and impairment of spatial memory retrieval. Importantly, no differences in general locomotor activity were observed as a function of SDS exposure.

This study does not include female voles in the experimental design.

We found that repeated SDS exposure, in male prairie voles, results in a depression-relevant phenotype resembling an anhedonia-like outcome (per reductions in sucrose preference) along with social withdrawal and spatial memory impairment - highlighting that the prairie vole is a valuable model with potential to study the neurobiology of social stress-induced depression-related outcomes.

Anxiety and depressive symptoms are common and highly interrelated. A relatively consistent temporal pattern of anxious and depressive symptoms has emerged from previous studies, such that the development of anxiety tends to precede and predict the development of depression rather than the other way around. Whether high levels of childhood anxiety predict depressive symptoms in late adolescence may depend, in part, on the ways in which children cope with stressful events. Accordingly, the present study used latent intercept models to examine involuntary and voluntary coping responses to familial stress as potential moderators of the association between childhood anxiety and adolescent depressive symptoms. Two hundred twenty-seven participants completed questionnaires measuring demographic variables as well as anxiety, depressive symptoms, and coping responses at a minimum of one time point over four waves of data collection (T1 Mage = 10.26 years, T2 Mage = 15.77 years, T3 Mage = 16.75 years, T4 Mage = 17.68 years). We found that childhood anxiety was positively associated with adolescent depressive symptoms when children reported higher levels of involuntary responses to family stress (e.g., rumination or physiological arousal) in conjunction with either lower levels of voluntary engaged responses (e.g., problem solving or emotion regulation) or higher levels of voluntary disengaged responses (e.g., avoidance or denial). These results shed light on the conditions under which childhood anxiety is associated with adolescent depressive symptoms and underscore the need for continued longitudinal and developmental research on this topic.

: Microtubule (MT) stability in neurons is vital for brain development; instability is associated with neuropsychiatric disorders. The present study examined the effects of social defeat stress (SDS) on MT-regulating proteins and tubulin polymerization.

: After 10 days of SDS, defeated mice were separated into susceptible (Sus) and unsusceptible (Uns) groups based on their performance in a social avoidance test. Using extracted brain tissues, we measured the expression levels of α-tubulin, acetylated α-tubulin, tyrosinated α-tubulin, MT-associated protein-2 (MAP2), stathmin (STMN1), phospho stathmin serine 16 (p-STMN1 [Ser16]), phospho stathmin serine 25 (p-STMN1 [Ser25]), phospho stathmin serine 38 (p-STMN1 [Ser38]), stathmin2 (STMN2), phospho stathmin 2 serine 73 (p-STMN2 [Ser73]), 78-kDa glucose-regulated protein (GRP-78), and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) using Western blot assay. The tubulin polymerization rate was also measured.

: We observed increased and decreased expression of acetylated and tyrosinated α-tubulin, respectively, decreased expression of p-STMN1 (Ser16) and increased expression of p-STMN1 (Ser25), p-STMN2 (Ser73) and GRP-78 and CHOP in the prefrontal cortex and/or hippocampus of defeated mice. A reduced tubulin polymerization rate was observed in the Sus group compared to the Uns and Con groups.

: Our findings suggest that SDS has detrimental effects on MT stability, and a lower tubulin polymerization rate could be a molecular marker for susceptibility to SDS.

Persistent stress increases the probability for developing depression significantly thereafter. Repeated social defeat stress is a widely used model to investigate depressive-like behavior in preclinical models. Hence, the repeated social defeat stress model provided an ideal animal model, through which the hypotheses of prevention and treatment can be investigated. We have successfully induced depressive-like behavior for male C57BL/6J mice with this model. Here, we reported that certain level of during-stress social interactions with single female or multiple male peer(s) exerted a positive role in preventing the development of depressive-like behavior induced by repeated social defeat stress. Our data suggested that the stress-susceptible mice may benefit from positive social interaction, which reduces the chance for depressive-like behavior development. Since numerous studies indicate that the metabotropic glutamate receptor 5 (mGluR5) plays an important role in various cognitive functions, we further investigate the treatment effect of 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) on the depressive-like behavior induced by repeated social defeat stress. Most importantly, robust anti-depressant effects have been achieved through modulating the mGluR5 function. We found that single oral dose administration of CDPPB (20 mg/kg), to some extent, alleviated the social avoidance behaviors for the stress-susceptible mice. Our data implies that the CDPPB, a positive allosteric modulator of mGluR5, is a promising anti-depressant candidate with limited side effect.

Studies in adults have linked stress-related activation of the immune system to the manifestation of psychiatric conditions. Using a translational design, this study aimed to examine the impact of social stress on immune activity in adolescents and on neuronal activity in a preclinical mouse model. Participants were 31 adolescents (ages 12-19), including 25 with mood and anxiety symptoms. Whole-blood samples were collected before and after the Trier Social Stress Test (TSST), a stress-inducing public speaking task, then cultured for 6 hours in the presence and absence of the inflammatory endotoxin lipopolysaccharide (LPS). Effects of TSST and LPS on 41 immune biomarkers were examined using repeated-measures analysis of variance. Separately, juvenile (8-week-old) male mice were non-stressed or exposed to reminder social defeat then intraperitoneally injected with saline or LPS (n = 6/group). Brains were perfused and collected for immunohistochemistry and confocal microscopy at 0, 1, 6, and 24 hours post-injection. Activity was determined by the density of cFos-positive neurons in the paraventricular hypothalamus, paraventricular thalamus, and basolateral amygdala, regions known to show sustained activation to immunological challenge. Analyses in the adolescent study indicated a strong effect of LPS but no effects of TSST or TSST×LPS interaction on immune biomarkers. Similarly, reminder social defeat did not induce sustained neuronal activity changes comparable to LPS immunological challenge in juvenile mice. Our convergent findings across species suggest that the acute immune response to stress documented in adults is not present in youth. Thus, aging and chronicity effects may play an important role in the inflammatory response to acute psychosocial stress.

Social defeat is a powerful experience leading to drastic changes in physiology and behavior, many of which are negative. For example, repeated social defeat in vertebrates results in reduced reproductive success, sickness and behavioral abnormalities that threaten individual survival and species persistence. However, little is known about what neural mechanisms are involved in determining whether an individual is resilient or susceptible to repeated social defeat stress. It also remains unknown whether exclusive use of reactive behaviors after repeated social defeat is maintained over time and impacts future behaviors during subsequent contests. We used a resident-intruder experiment in the African cichlid fish Astatotilapia burtoni to investigate the behavior and neural correlates of these two opposing groups. Behavior was quantified by watching fish during defeat trials and used to distinguish resilient and susceptible individuals. Both resilient and susceptible fish started with searching and freezing behaviors, with searching decreasing and freezing increasing after repeated social defeat. After a 4 day break period, resilient fish used both searching and freezing behaviors during a social defeat encounter with a new resident, while susceptible fish almost exclusively used freezing behaviors. By quantifying neural activation using pS6 in socially relevant brain regions, we identified differential neural activation patterns associated with resilient and susceptible fish and found nuclei that co-varied and may represent functional networks. These data provide the first evidence of specific conserved brain networks underlying social stress resilience and susceptibility in fishes.

Exposure to acute and chronic stress has significant effects on the basic mechanisms of associative learning and memory. Stress can both impair and enhance associative learning depending on type, intensity, and persistence of the stressor, the subject's sex, the context that the stress and behavior is experienced in, and the type of associative learning taking place. In some cases, stress can cause or exacerbate the maladaptive behavior that underlies numerous psychiatric conditions including anxiety disorders, obsessive-compulsive disorder, post-traumatic stress disorder, substance use disorder, and others. Therefore, it is critical to understand how the varied effects of stress, which may normally facilitate adaptive behavior, can also become maladaptive and even harmful. In this review, we highlight several findings of associative learning and decision-making processes that are affected by stress in both human and non-human subjects and how they are related to one another. An emerging theme from this work is that stress biases behavior towards less flexible strategies that may reflect a cautious insensitivity to changing contingencies. We consider how this inflexibility has been observed in different associative learning procedures and suggest that a goal for the field should be to clarify how factors such as sex and previous experience influence this inflexibility.

Neuropsychiatric disorders, such as major depression, anxiety disorders, and post-traumatic stress disorder, tend to be long-term conditions in whose development and maintenance stress are central pathogenic factors. Translational mouse models are widely used in neuropsychiatric research, exploiting social and non-social stressors to investigate the mechanisms underlying their detrimental effects. However, most studies focus on the short-term consequences of chronic stress, whereas only a few are interested in the long-term course. This is counterintuitive given the human conditions that preclinical models are designed to mimic. In this review, we have summarized the limited work to date on long-term effects of chronic stress in mice models. First, the different models are presented and a definition of short- vs. long-term sequelae is proposed. On this basis, behavioral, endocrine, and vegetative effects are addressed before examining data on cellular and molecular alterations in the brain. Finally, future directions for research on the long-term effects of stress are discussed.

Differential expression of myelin-related genes and changes in myelin thickness have been demonstrated in mice after chronic psychosocial stress, a risk factor for anxiety disorders. To determine whether and how stress affects structural remodeling of nodes of Ranvier, another form of myelin plasticity, we developed a 3D reconstruction analysis of node morphology in C57BL/6NCrl and DBA/2NCrl mice. We identified strain-dependent effects of chronic social defeat stress on node morphology in the medial prefrontal cortex (mPFC) gray matter, including shortening of paranodes in C57BL/6NCrl stress-resilient and shortening of node gaps in DBA/2NCrl stress-susceptible mice compared to controls. Neuronal activity has been associated with changes in myelin thickness. To investigate whether neuronal activation is a mechanism influencing also node of Ranvier morphology, we used DREADDs to repeatedly activate the ventral hippocampus-to-mPFC pathway. We found reduced anxiety-like behavior and shortened paranodes specifically in stimulated, but not in the nearby non-stimulated axons. Altogether, our data demonstrate (1) nodal remodeling of the mPFC gray matter axons after chronic stress and (2) axon-specific regulation of paranodes in response to repeated neuronal activity in an anxiety-associated pathway. Nodal remodeling may thus contribute to aberrant circuit function associated with anxiety disorders.

Severe stress exposure increases the risk of stress-related disorders such as major depressive disorder (MDD). An essential characteristic of MDD is the impairment of social functioning and lack of social motivation. Chronic social defeat stress is an established animal model for MDD research, which induces a cascade of physiological and behavioral changes. Current markerless pose estimation tools allow for more complex and naturalistic behavioral tests. Here, we introduce the open-source tool DeepOF to investigate the individual and social behavioral profile in mice by providing supervised and unsupervised pipelines using DeepLabCut-annotated pose estimation data. Applying this tool to chronic social defeat in male mice, the DeepOF supervised and unsupervised pipelines detect a distinct stress-induced social behavioral pattern, which was particularly observed at the beginning of a novel social encounter and fades with time due to habituation. In addition, while the classical social avoidance task does identify the stress-induced social behavioral differences, both DeepOF behavioral pipelines provide a clearer and more detailed profile. Moreover, DeepOF aims to facilitate reproducibility and unification of behavioral classification by providing an open-source tool, which can advance the study of rodent individual and social behavior, thereby enabling biological insights and, for example, subsequent drug development for psychiatric disorders.

Adverse early life experiences affect neuronal growth and maturation of reward circuits that modify behavior under reward predicting conditions. Previous studies demonstrate that rats undergoing denial of expected reward in the form of maternal contact (DER-animal model of maternal neglect) during early post-natal life developed anhedonia, aggressive play-fight behaviors and aberrant prefrontal cortex structure and neurochemistry. Although many studies revealed social deficiency following early-life stress most reports focus on individual animal tasks. Thus, attention needs to be given on the social effects during group tasks in animals afflicted by early life adversity.

To investigate the potential impact of the DER experience on the manifestation of behavioral responses induced by natural rewards, we evaluated: 1) naïve adult male sexual preference and performance, and 2) anticipatory behavior during a group 2-phase food anticipation learning task composed of a context-dependent and a cue-dependent learning period.

DER rats efficiently spent time in the vicinity of and initiated sexual intercourse with receptive females suggesting an intact sexual reward motivation and consummation. Interestingly, during the context-dependent phase of food anticipation training DER rats displayed a modified exploratory activity and lower overall reward-context association. Moreover, during the cue-dependent phase DER rats displayed a mild deficit in context-reward association while increased cue-dependent locomotion. Additionally, DER rats displayed unstable food access priority following food presentation. These abnormal behaviours were accompanied by overactivation of the ventral prefrontal cortex and nucleus accumbens, as assessed by pCREB levels.

Collectively, these data show that the neonatal DER experience resulted in adulthood in altered activation of the reward circuitry, interfered with the normal formation of context-reward associations, and disrupted normal reward access hierarchy formation. These findings provide additional evidence to the deleterious effects of early life adversity on reward system, social hierarchy formation, and brain function.

Chronic social defeat has been found to be stressful and to affect many aspects of the brain and behaviors in males. However, relatively little is known about its effects on females. In the present study, we examined the effects of repeated social defeat on social approach and anxiety-like behaviors as well as the neuronal activation in the brain of sexually naïve female Mongolian gerbils (Meriones unguiculatus). Our data indicate that repeated social defeats for 20 days reduced social approach and social investigation, but increased risk assessment or vigilance to an unfamiliar conspecific. Such social defeat experience also increased anxiety-like behavior and reduced locomotor activity. Using ΔFosB-immunoreactive (ΔFosB-ir) staining as a marker of neuronal activation in the brain, we found significant elevations by social defeat experience in the density of ΔFosB-ir stained neurons in several brain regions, including the prelimbic (PL) and infralimbic (IL) subnuclei of the prefrontal cortex (PFC), CA1 subfields (CA1) of the hippocampus, central subnuclei of the amygdala (CeA), the paraventricular nucleus (PVN), dorsomedial nucleus (DMH), and ventrolateral subdivision of the ventromedial nucleus (VMHvl) of the hypothalamus. As these brain regions have been implicated in social behaviors and stress responses, our data suggest that the specific patterns of neuronal activation in the brain may relate to the altered social and anxiety-like behaviors following chronic social defeat in female Mongolian gerbils.

Emerging evidence from animal and human studies has suggested that small microbial metabolites generated in the gut influence host mood and behavior. Our previous study reported that patients with major depressive disorder (MDD) reduced the abundance of genera Blautia and Eubacterium, the microbials critically regulating cholesterol and bile acid metabolism in the gut. In this study, we further demonstrated that the levels of plasma bile acid chenodeoxycholic acid (CDCA) were significantly lower in Chinese MDD patients (142) than in healthy subjects (148). Such low levels of plasma CDCA in MDD patients were rescued in remitters but not in nonremitters following antidepressant treatment. In a parallel animal study, Chronic Social Defeat Stress (CSDS) depressed mice reduced the plasma CDCA and expression level in prefrontal cortex (PFC) of bile acid receptor (FXR) protein, which is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. We found that CDCA treatment restored the level of FXR in the CSDS mice, suggesting the involvement of bile acid receptors in MDD. We observed that CDCA decreased the activity of the NLRP3 inflammasome and caspase-1 and subsequently increased the levels of phosphorylation and expression of PFC glutamate receptors (GluA1) in the PFC. In addition, CDCA showed antidepressant effects in the tests of sucrose preference, tail suspension, and forced swimming in CSDS mouse model of depression. Finally, in agreement with this idea, blocking these receptors by a FXR antagonist GS abolished CDCA-induced antidepressant effect. Moreover, CDCA treatment rescued the increase of IL-1β, IL-6, TNF α and IL-17, which also were blocked by GS. These results suggest that CDCA is a biomarker and target potentially important for the diagnosis and treatment of MDD.

Laboratory mouse models offer opportunities to bridge the gap between basic neuroscience and applied stress research. Here we consider the ecological validity of social defeat stressors in mouse models of emotional vulnerability and resilience. Reports identified in PubMed from 1980 to 2020 are reviewed for the ecological validity of social defeat stressors, sex of subjects, and whether results are discussed in terms of vulnerability alone, resilience alone, or both vulnerability and resilience. Most of the 318 reviewed reports (95%) focus on males, and many reports (71%) discuss vulnerability and resilience. Limited ecological validity is associated with increased vulnerability and decreased resilience. Elements of limited ecological validity include frequent and repeated exposure to defeat stressors without opportunities to avoid or escape from unfamiliar conspecifics that are pre-screened and selected for aggressive behavior. These elements ensure defeat and may be required to induce vulnerability, but they are not representative of naturalistic conditions. Research aimed at establishing causality is needed to determine whether ecologically valid stressors build resilience in both sexes of mice.

Depressive disorders are the most burdensome psychiatric disorders worldwide. Although huge efforts have been made to advance treatment, outcomes remain unsatisfactory. Many factors contribute to this gridlock including suboptimal animal models. Especially limited study comparability and replicability due to imprecise terminology concerning depressive-like states are major problems. To overcome these issues, new approaches are needed. Here, we introduce a taxonomical concept for modelling depression in laboratory mice, which we call depression-like syndrome (DLS). It hinges on growing evidence suggesting that mice possess advanced socioemotional abilities and can display non-random symptom patterns indicative of an evolutionary conserved disorder-like phenotype. The DLS approach uses a combined heuristic method based on clinical depression criteria and the Research Domain Criteria to provide a biobehavioural reference syndrome for preclinical rodent models of depression. The DLS criteria are based on available, species-specific evidence and are as follows: (I) minimum duration of phenotype, (II) significant sociofunctional impairment, (III) core biological features, (IV) necessary depressive-like symptoms. To assess DLS presence and severity, we have designed an algorithm to ensure statistical and biological relevance of findings. The algorithm uses a minimum combined threshold for statistical significance and effect size (p value ≤ 0.05 plus moderate effect size) for each DLS criterion. Taken together, the DLS is a novel, biologically founded, and species-specific minimum threshold approach. Its long-term objective is to gradually develop into an inter-model validation standard and microframework to improve phenotyping methodology in translational research.