Focus: The Science of Stress: The Impact of Maternal Antioxidants on Prenatal Stress Effects on Offspring Neurobiology and Behavior

Prenatal stress is a neuropsychiatric risk factor, and effects may be mediated by prenatal oxidative stress. Cell types in the brain sensitive to oxidative stress—cortical microglia and cortical and hippocampal interneurons—may be altered by oxidative stress generated during prenatal stress and may be neurobiological substrates for altered behavior. Our objective was to determine the critical nature of oxidative stress in prenatal stress effects by manipulating prenatal antioxidants. CD1 mouse dams underwent restraint embryonic day 12 to 18 three times daily or no stress and received intraperitoneal injections before each stress period of vehicle, N-acetylcysteine (200 mg/kg daily), or astaxanthin (30 mg/kg before first daily stress, 10 mg/kg before second/third stresses). Adult male and female offspring behavior, microglia, and interneurons were assessed. Results supported the hypothesis that prenatal stress-induced oxidative stress affects microglia; microglia ramification increased after prenatal stress, and both antioxidants prevented these effects. In addition, N-acetylcysteine or astaxanthin was effective in preventing distinct male and female interneuron changes; decreased female medial frontal cortical parvalbumin interneurons was prevented by either antioxidant; increased male medial frontal cortical parvalbumin interneurons was prevented by N-acetylcysteine and decreased male hippocampal GAD67GFP+ cells prevented by astaxanthin. Prenatal stress-induced increased anxiety-like behavior and decreased sociability were not prevented by prenatal antioxidants. Sensorimotor gating deficits in males was partially prevented by prenatal astaxanthin. This study demonstrates the importance of oxidative stress for persistent impacts on offspring cortical microglia and interneurons, but did not link these changes with anxiety-like, social, and sensorimotor gating behaviors.     

Deficiency of antinociception and excessive grooming induced by acute immobilization stress in Per1 mutant mice

Acute stressors induce changes in numerous behavioral parameters through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Several important hormones in paraventricular nucleus of the hypothalamus (PVN) play the roles in these stress-induced reactions. Corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and corticosterone are considered as molecular markers for stress-induced grooming behavior. Oxytocin in PVN is an essential modulator for stress-induced antinociception. The clock gene, Per1, has been identified as an effecter response to the acute stresses, but its function in neuroendocrine stress systems remains unclear. In the present study we observed the alterations in grooming and nociceptive behaviors induced by acute immobilization stress in Per1 mutant mice and other genotypes (wild types and Per2 mutant). The results displayed that stress elicited a more robust effect on grooming behavior in Per1 mutant mice than in other genotypes. Subsequently, the obvious stress-induced antinociception was observed in the wild-type and Per2 mutant mice, however, in Per1 mutant, this antinociceptive effects were partially-reversed (mechanical sensitivity), or over-reversed to hyperalgesia (thermal sensitivity). The real-time qPCR results showed that in PVN, there were stress-induced up-regulations of Crh, Avp and c-fos in all of genotypes; moreover, the expression change of Crh in Per1 mutant mice was much larger than in others. Another hormonal gene, Oxt, was up-regulated induced by stress in wild-type and Per2 mutant but not in Per1 mutant. In addition, the stress significantly elevated the serum corticosterone levels without genotype-dependent differences, and accordingly the glucocorticoid receptor gene, Nr3c1, expressed with a similar pattern in PVN of all strains. Taken together, the present study indicated that in acute stress treated Per1 mutant mice, there are abnormal hormonal responses in PVN, correlating with the aberrant performance of stress-induced behaviors. Therefore, our findings suggest a novel functional role of Per1 in neuroendocrine stress system, which further participates in analgesic regulation.     

Effect of LPS treatment on tyrosine hydroxylase expression and Parkinson-like behaviors

Puberty is a critical period of development during which the brain undergoes reorganizing and remodeling. Exposure to stress during this period is thought to interfere with normal brain development and increase susceptibility to mental illnesses. In female mice, pubertal exposure to lipopolysaccharide (LPS), a bacterial endotoxin, has been shown to alter sexual, anxiety-like, and depression-like behaviors and cognition in an enduring manner. However, the mechanisms underlying these effects remain unknown. The present study examined age and sex difference in tyrosine hydroxylase (TH) expression and dopamine-dependent and Parkinson-like behaviors following LPS treatment. The results show that LPS treatment during adulthood causes an enduring increase in TH expression in many of the brain regions examined. In contrast, there is no change in TH expression following LPS treatment during puberty. However, pubertal LPS treatment induces enduring behavioral deficits in tests of Parkinson-like behaviors, more so in male than in female mice. These results suggest that the low levels of TH following exposure to pubertal immune challenge may predispose mice to Parkinson-like behavior. These findings add to our understanding of stress and immune responses during puberty and their impact on mental health later in life.     

Prenatal Stress Impairs Spinal Cord Oligodendrocyte Maturation via BDNF Signaling in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis

One of the most substantial and established environmental risk factors for neurological and psychiatric disorders is stress exposure, whose detrimental consequences hinge on several variables including time. In this regard the gestational period is known to present an intrinsic vulnerability to environmental insults and thus stressful events during pregnancy can lead to severe consequences on the offspring’s brain development with long-term repercussions throughout adulthood. On this basis, we investigated the long-lasting impact of prenatal stress exposure on the susceptibility to the experimental autoimmune encephalomyelitis (EAE), a well-established murine model of multiple sclerosis. Although stress is considered a triggering factor for this chronic, progressive, autoimmune disease, little is known about the underlying mechanisms. To this end, EAE was induced by immunization with MOG35-55/CFA and pertussis toxin administration in adult female C57BL/6 mice born from control or stressed dams exposed to restraint stress during the last days of gestation. Our results demonstrate that gestational stress induces a marked increase in the severity of EAE symptoms in adulthood. Further, we highlight an altered maturation of oligodendrocytes in the spinal cord of prenatally stressed EAE mice, as indicated by the higher levels of GPR17, a marker of immature oligodendrocyte precursor cells. These behavioral and molecular alterations are paralleled by changes in the expression and signaling of the neurotrophin BDNF, an important mediator of neural plasticity that may contribute to stress-induced impaired remyelination. Since several already marketed drugs are able to modulate BDNF levels, these results pave the way to the possibility of repositioning these drugs in multiple sclerosis.

     

Development of trophic interactions in the vertebrate peripheral nervous system

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of thetrk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.     

Stress, cognitive impairment and cell adhesion molecules

Stress has profound effects on brain structure and function, but the underlying mechanisms are still poorly understood. Recent studies imply that neuronal cell adhesion molecules of the immunoglobulin superfamily--NCAM and L1--are important mediators of the effects of stress on the brain. Chronic stress regimes that lead to hippocampal atrophy and spatial-learning impairment in rodents simultaneously induce a pattern of changes in cell adhesion molecule expression that fits with a role for these molecules in stress-induced neuronal damage and neuroprotective mechanisms. These findings highlight cell adhesion molecules as potential therapeutic targets to treat stress-related cognitive disturbances.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

Modulation of monoamine transporter expression and function by repetitive transcranial magnetic stimulation

Repetitive transcranial magnetic stimulation (rTMS) is a new tool for the treatment of neuropsychiatric disorders. However, the mechanisms underlying the effects of rTMS are still unclear. In this study, we analyzed mRNA expression changes of monoamine transporter (MAT) genes, which are targets for antidepressants and psychostimulants. Following a 20-day rTMS treatment, these genes were found to be differentially expressed in the mouse brain. Down-regulation of serotonin transporter (SERT) mRNA levels and the subsequent decrease in serotonin uptake and binding were observed after chronic rTMS. In contrast to the SERT changes, increased mRNA levels of dopamine transporter (DAT) and norepinephrine transporter (NET) were observed. For NET, but not DAT, there were accompanying changes in uptake and binding. Similar effect on NET was observed in PC12 cells stimulated by rTMS for 15 days. These results indicate that modulation of MATs by chronic rTMS may be one therapeutic mechanism for the treatment of neuropsychiatric disorders.     

"Green odor" inhalation reduces the skin-barrier disruption induced by chronic restraint stress in rats: physiological and histological examinations

We investigated whether inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) prevents the skin-barrier disruption induced by chronic restraint stress in rats. To this end, transepidermal water loss (TEWL) was measured as an index of the disruption of skin-barrier function, whereas light- and electron-microscope examinations were performed to observe histological changes in the skin of the stressed animals. In addition, the effects on TEWL induced by chronic administration of a glucocorticoid, dexamethasone (DEX), were examined. Chronic restraint stress (8 h per day for 14 days) increased TEWL (vehicle + stress group). This effect (and the chronic stress-induced increase in adrenal weight) was prevented in rats that inhaled green odor at the beginning of each day's restraint (2 h each day for 14 days; green odor + stress group). Electron-microscope studies revealed that rats in the green odor + stress group possessed sufficient intercorneocyte lipids to create an effective skin barrier, although these had apparently been decreased in the vehicle + stress group. Daily administration of DEX for 14 days increased TEWL. The present results suggest that chronic stress-induced disruption of the skin barrier in rats can be reduced or prevented by green odor (possibly at least in part through an inhibitory effect on the stress-induced activation of the hypothalamo-pituitary-adrenocortical axis).     

Effects of chronic mild stress on behavioral and neurobiological parameters — Role of glucocorticoid

Major depression is thought to originate from maladaptation to adverse events, particularly when impairments occur in mood-related brain regions. Hypothalamus–pituitary–adrenal (HPA) axis is one of the major systems involved in physiological stress response. HPA axis dysfunction and high glucocorticoid concentrations play an important role in the pathogenesis of depression. In addition, astrocytic disability and dysfunction of neurotrophin brain-derived neurotrophin factor (BDNF) greatly influence the development of depression and anxiety disorders. Therefore, we investigated whether depressive-like and anxiety-like behaviors manifest in the absence of glucocorticoid production and circulation in adrenalectomized (ADX) rats after chronic mild stress (CMS) exposure and its potential molecular mechanisms. The results demonstrate that glucocorticoid-controlled rats showed anxiety-like behaviors but not depression-like behaviors after CMS. Molecular and cellular changes included the decreased BDNF in the hippocampus, astrocytic dysfunction with connexin43 (cx43) decreasing and abnormality in gap junction in prefrontal cortex (PFC). Interestingly, we did not find any changes in glucocorticoid receptor (GR) or its chaperone protein FK506 binding protein 51 (FKBP5) expression in the hippocampus or PFC in ADX rats subjected to CMS. In conclusion, the production and circulation of glucocorticoids are one of the contributing factors in the development of depression-like behaviors in response to CMS. In contrast, the effects of CMS on anxiety-like behaviors are independent of the presence of circulating glucocorticoids. Meanwhile, stress decreased GR expression and enhanced FKBP5 expression via higher glucocorticoid exposure. Gap junction dysfunction and changes in BDNF may be associated with anxiety-like behaviors.     

Cathepsin C Aggravates Neuroinflammation Involved in Disturbances of Behaviour and Neurochemistry in Acute and Chronic Stress-Induced Murine Model of Depression

Major depression has been interpreted as an inflammatory disease characterized by cell-mediated immune activation, which is generally triggered by various stresses. Microglia has been thought to be the cellular link between inflammation and depression-like behavioural alterations. The expression of cathepsin C (Cat C), a lysosomal proteinase, is predominantly induced in microglia in neuroinflammation. However, little is known about the role of Cat C in pathophysiology of depression. In the present study, Cat C transgenic mice and wild type mice were subjected to an intraperitoneal injection of LPS (0.5 mg/kg) and 6-week unpredictable chronic mild stress (UCMS) exposure to establish acute and chronic stress-induced depression model. We examined and compared the behavioural and proinflammatory cytokine alterations in serum and depression-targeted brain areas of Cat C differentially expressed mice in stress, as well as indoleamine 2,3-dioxygenase (IDO) and 5-hydroxytryptamine (5HT) levels in brain. The results showed that Cat C overexpression (Cat C OE) promoted peripheral and central inflammatory response with significantly increased TNFα, IL-1β and IL-6 in serum, hippocampus and prefrontal cortex, and resultant upregulation of IDO and downregulation of 5HT expression in brain, and thereby aggravated depression-like behaviours accessed by open field test, forced swim test and tail suspension test. In contrast, Cat C knockdown (Cat C KD) partially prevented inflammation, which may help alleviate the symptoms of depression in mice. To the best of our knowledge, we are the first to demonstrate that Cat C aggravates neuroinflammation involved in disturbances of behaviour and neurochemistry in acute and chronic stress-induced murine model of depression.     

CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Fucoidan exerts antidepressant-like effects in mice via regulating the stability of surface AMPARs

The inflammatory hypothesis is one of the most important mechanisms of depression. Fucoidan is a bioactive sulfated polysaccharide abundant in brown seaweeds with anti-inflammatory activity. However, the antidepressant effects of fucoidan on chronic stress-induced depressive-like behaviors have not been well elucidated. Here, we used two different depressive-like mouse models, lipopolysaccharide (LPS) and chronic restraint stress (CRS) models, to explore the detailed molecular mechanism underlying its antidepressant-like effects in C57BL/6J mice by combining multiple behavioral, molecular and immunofluorescence experiments. Adenovirus-mediated overexpression of caspase-1 and pharmacological inhibitors were also used to clarify the antidepressant mechanisms of fucoidan. We found that acute administration of fucoidan did not produce antidepressant effects in the tail suspension test (TST) and forced swim test (FST). Interestingly, chronic fucoidan administration not only dose-dependently reduced stress-induced depressive-like behaviors in the TST, FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT), but also alleviated the downregulation of brain-derived neurotrophic factor (BDNF)-dependent synaptic plasticity via inhibiting caspase-1-mediated inflammation in the hippocampus of mice. Moreover, fucoidan significantly ameliorated behavioral and synaptic plasticity abnormalities in the overexpression of caspase-1 in the hippocampus of mice. Furthermore, blocking BDNF abolished the antidepressant-like effects of fucoidan in mice. Therefore, our findings clearly indicate that fucoidan provides a potential supplementary noninvasive treatment for depression by inhibition of hippocampal inflammation.     

Interactions of norepinephrine and galanin in the central amygdala and lateral bed nucleus of the stria terminalis modulate the behavioral response to acute stress

Many aspects of drug abuse and addiction share neurobiological substrates with the modulatory processes underlying the response and adaptation to acute stress. In particular, the ascending noradrenergic system has been implicated in facilitating the response to stress, and in stress-induced reinstatement of drug seeking behavior. Thus, to better understand the link between stress and addictive behaviors, it would be informative to understand better the modulatory function of the ascending noradrenergic system, and its interaction with other neurotransmitters with which it is closely associated or co-localized, such as the neuropeptide galanin. In this paper, we review a series of studies investigating the functional interactions of norepinephrine and galanin in modulating the behavioral response to acute stress in two components of the extended amygdala, the central nucleus of the amygdala and the lateral bed nucleus of the stria terminalis. We showed that norepinephrine facilitates behavioral reactivity to stress on the elevated plus-maze and social interaction tests. However, when stress-induced activation of the noradrenergic system was enhanced by blocking inhibitory adrenergic autoreceptors, galanin release was recruited in the central amygdala, acting to attenuate the behavioral response to stress. By contrast, stress-induced galanin release in the lateral bed nucleus appeared to be independent of enhanced noradrenergic activation, and unlike the central amygdala, both galanin and norepinephrine facilitated behavioral stress reactivity in the bed nucleus. The different modes of interaction and differential region- and response-specificity of galanin and norepinephrine suggest that a complex neural circuit interconnecting these two regions is involved in the modulatory effects of norepinephrine and galanin on the behavioral response to stress. Such complexity may allow for flexibility and plasticity in stress adaptation, and may also contribute to behavioral changes induced by chronic drug administration. Thus, the interaction of galanin and norepinephrine may be a viable target for the future development of novel therapeutic strategies for treating behavioral disorders related to stress or drug abuse.     

Acute and Chronic Administration of the Branched-Chain Amino Acids Decreases Nerve Growth Factor in Rat Hippocampus

Maple syrup urine disease (MSUD) is a neurometabolic disorder caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA) and their corresponding branched-chain α-keto acids. In this study, we examined the effects of acute and chronic administration of BCAA on protein levels and mRNA expression of nerve growth factor (NGF) considering that patients with MSUD present neurological dysfunction and cognitive impairment. Considering previous observations, it is suggested that oxidative stress may be involved in the pathophysiology of the neurological dysfunction of MSUD. We also investigated the influence of antioxidant treatment (N-acetylcysteine and deferoxamine) in order to verify the influence of oxidative stress in the modulation of NGF levels. Our results demonstrated decreased protein levels of NGF in the hippocampus after acute and chronic administration of BCAA. In addition, we showed a significant decrease in the expression of ngf in the hippocampus only following acute administration in 10-day-old rats. Interestingly, antioxidant treatment was able to prevent the decrease in NGF levels by increasing ngf expression. In conclusion, the results suggest that BCAA is involved in the regulation of NGF in the developing rat. Thus, it is possible that alteration of neurotrophin levels during brain maturation could be of pivotal importance in the impairment of cognition provoked by BCAA. Moreover, the decrease in NGF levels was prevented by antioxidant treatment, reinforcing that the hypothesis of oxidative stress can be an important pathophysiological mechanism underlying the brain damage observed in MSUD.     

Regular Moderate or Intense Exercise Prevents Depression-Like Behavior without Change of Hippocampal Tryptophan Content in Chronically Tryptophan-Deficient and Stressed Mice

Regular exercise has an antidepressant effect in human subjects. Studies using animals have suggested that the antidepressant effect of exercise is attributable to an increase of brain 5-hydroxytryptamine (5-HT); however, the precise mechanism underlying the antidepressant action via exercise is unclear. In contrast, the effect of 5-HT on antidepressant activity has not been clarified, in part because the therapeutic response to antidepressant drugs has a time lag in spite of the rapid increase of brain 5-HT upon administration of these drugs. This study was designed to investigate the contribution of brain 5-HT to the antidepressant effect of exercise. Mice were fed a tryptophan-deficient diet and stressed using chronic unpredictable stress (CUS) for 4 weeks with or without the performance of either moderate or intense exercise on a treadmill 3 days per week. The findings demonstrated that the onset of depression-like behavior is attributable not to chronic reduction of 5-HT but to chronic stress. Regular exercise, whether moderate or intense, prevents depression-like behavior with an improvement of adult hippocampal cell proliferation and survival and without the recovery of 5-HT. Concomitantly, the mice that exercised showed increased hippocampal noradrenaline. Regular exercise prevents the impairment of not long-term memory but short-term memory in a 5-HT-reduced state. Together, these findings suggest that: (1) chronic reduction of brain 5-HT may not contribute to the onset of depression-like behavior; (2) regular exercise, whether moderate or intense, prevents the onset of chronic stress-induced depression-like behavior independent of brain 5-HT and dependent on brain adrenaline; and (3) regular exercise prevents chronic tryptophan reduction-induced impairment of not long-term but short-term memory.     

Neurotrophins as mediators of drug effects on mood, addiction, and neuroprotection

The induction of synthesis or release of endogenous neurotrophic factors in the brain by low-molecular-weight drugs could be a feasible alternative for the direct administration of neurotrophic factors for the treatment of central nervous system disorders. Recent data suggest that several drugs already in clinical use increase the synthesis, release, or signaling of neurotrophins. Antidepressant drugs increase the synthesis and signaling of brain-derived neurotrophic factor (BDNF), and BDNF signaling appears to be both sufficient and necessary for the antidepressant-induced behavioral effects. Furthermore, neurotrophins and other neurotrophic factors play a role in the acute and chronic responses produced by addictive drugs. Moreover, several neuroprotective drugs influence neurotrophin synthesis or signaling, although the significance of these effects is still unclear. These findings reveal a wider role for neurotrophic factors in drug action than has previously been expected, and they suggest that neurotrophin-induced trophic responses in neuronal connectivity and plasticity may be involved in the mechanism of action of several classes of CNS drugs. Improved assay systems are needed for the systematic screening of the effects of putative neuroprotective drugs on the synthesis, release, and signaling of neurotrophic factors, and for the evaluation of the functional role of these factors in the action of novel drug candidates.     

Enkephalin knockout male mice are resistant to chronic mild stress

Enhanced stress reactivity or sensitivity to chronic stress increases the susceptibility to mood pathologies such as major depression. The opioid peptide enkephalin is an important modulator of the stress response. Previous studies using preproenkephalin knockout (PENK KO) mice showed that these animals exhibit abnormal stress reactivity and show increased anxiety behavior in acute stress situations. However, the consequence of enkephalin deficiency in the reactivity to chronic stress conditions is not known. In this study, we therefore submitted wild‐type (WT) and PENK KO male mice to chronic stress conditions, using the chronic mild stress (CMS) protocol. Subsequently, we studied the CMS effects on the behavioral and hormonal level and also performed gene expression analyses. In WT animals, CMS increased the expression of the enkephalin gene in the paraventricular nucleus (PVN) of the hypothalamus and elevated the corticosterone levels. In addition, WT mice exhibited enhanced anxiety in the zero‐maze test and depression‐related behaviors in the sucrose preference and forced swim tests. Surprisingly, in PENK KO mice, we did not detect anxiety and depression‐related behavioral changes after the CMS procedure, and even measured a decreased hormonal stress response. These results indicate that PENK KO mice are resistant to the CMS effects, suggesting that enkephalin enhances the reactivity to chronic stress.