Maternal Separation Rats Research Articles

Early-Life Stress Influences the Transcriptional Activation of Alpha-2A Adrenergic Receptor and Associated Protein Kinase A Signaling Molecules in the Frontal Cortex of Rats.

Early life is a highly sensitive period associated with profound changes in brain structure and function. Adverse experiences of early-life stress (ELS) are prominent risk factors for the precipitation of major depressive disorder (MDD). In recent years, dysfunction of the central noradrenergic (NA) system and subsequent deficits in norepinephrine (NE) signaling have gained increasing attention in the pathophysiology of MDD. However, the role of the α-2A adrenergic receptor and its downstream second messenger signaling system has not been investigated in connection to early-life stress-induced depression, limiting valuable insights into neurobiological mechanisms underlying this disorder. In this study, we used maternal separation (MS) as a rodent model of ELS to investigate whether ELS-induced depressive behavior is related to the α-2A adrenergic receptor and its associated second messenger signaling cascade. To do so, we studied expression levels of the α-2A adrenergic receptor (Adra2a), G alpha proteins (stimulatory subunit-Gαs [Gnas] and inhibitory subunit-Gαi [Gnai1 and Gnai2]), and downstream protein kinase A (PKA) catalytic [Prkarcα and Prkarcβ] and regulatory subunits [Prkar1α, Prkar1β, Prkar2α, and Prkar2β]) in the frontal cortex (FC) of MS rats. We found reduced sucrose preference in MS animals, along with reduced transcript levels of Adra2a, Gnai2, Prkar1β, and Prkarcβ. These findings suggest that ELS exposure may contribute to depression symptomatology via alterations in the expression of key genes involved in the NA system, highlighting potential mechanisms underlying ELS-induced depressive behavior.

Docosahexaenoic acid ameliorates autistic‐like behaviors by inhibiting oxidative stress and inflammatory response in neonatal maternal separation rats

AbstractAutism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impaired social interactions and communication, repetitive, or stereotyped behavior. Docosahexaenoic acid (DHA), an essential polyunsaturated fatty acid, has been demonstrated to exert anti‐oxidative stress and anti‐inflammatory properties, while also promoting myelin development and neural differentiation and development. However, it remains uncertain whether DHA can ameliorate autistic‐like behaviors, and if so, the underlying mechanisms are still unclear. Here, we established a neonatal maternal separation (NMS) rat model and treated it with DHA (80 mg/kg/day, i.p.). The results showed DHA treatment significantly alleviated autism‐like behaviors in the NMS rats during their juvenile period. Subsequently, we employed network pharmacology analysis and molecular docking methods to screen potential targets of DHA in ASD therapy. Through Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Wikipedia enrichment analysis, we identified anti‐oxidative stress, anti‐inflammatory and JNK signaling pathway that might be associated with DHA‐mediated improvement of autistic‐like behaviors. Furthermore, western blotting assays showed DHA significantly downregulated the expression levels of p‐JNK and c‐JUN, while upregulating the expression levels of NRF2, HO‐1, SOD1, and CAT. In addition, enzyme‐linked immunosorbent assay results showed DHA effectively reduced the production of pro‐inflammatory factors, such as TNF‐α, IL‐6, and IL‐1β. Collectively, our study predicted and validated that DHA exhibits the potential to improve autistic‐like behaviors induced by NMS in rats by suppressing JNK activation and inhibiting oxidative stress and inflammatory response. These findings suggest that DHA may be a potential therapeutic agent for the treatment of ASD.

Boosting decision-making in rat models of early-life adversity with environmental enrichment and intranasal oxytocin

Impaired decision-making constitutes a fundamental issue in numerous psychiatric disorders. Extensive research has established that early life adversity (ELA) increases vulnerability to psychiatric disorders later in life. ELA in human neonates is associated with changes in cognitive, emotional, as well as reward-related processing. Maternal separation (MS) is an established animal model of ELA and has been shown to be associated with decision-making deficits. On the other hand, enriched environment (EE) and intranasal oxytocin (OT) administration have been demonstrated to have beneficial effects on decision-making in humans or animals. Given these considerations, our investigation sought to explore the impact of brief exposure to EE and intranasal OT administration on the decision-making abilities of adolescent rats that had experienced MS during infancy.The experimental protocol involved subjecting rat pups to the MS regimen for 180 min per day from postnatal day (PND) 1 to PND 21. Then, from PND 22 to PND 34, the rats were exposed to EE and/or received intranasal OT (2 μg/μl) for seven days. The assessment of decision-making abilities, using a rat gambling task (RGT), commenced during adolescence. Our findings revealed that MS led to impaired decision-making and a decreased percentage of advantageous choices. However, exposure to brief EE or intranasal OT administration mitigated the deficits induced by MS and improved the decision-making skills of maternally-separated rats. Furthermore, combination of these treatments did not yield additional benefits. These results suggest that EE and OT may hold promise as therapeutic interventions to enhance certain aspects of cognitive performance.

Paeoniflorin Alleviates Anxiety and Visceral Hypersensitivity via HPA Axis and BDNF/TrkB/PLCγ1 Pathway in Maternal Separation-induced IBS-like Rats.

Irritable Bowel Syndrome (IBS) is a prevalent gastrointestinal disorder that significantly diminishes the quality of life for affected individuals. The pathophysiology of IBS remains poorly understood, and available therapeutic options for IBS are limited. The crucial roles of brain-gut interaction, which is mediated by the Hypothalamic-Pituitary-Adrenocortical (HPA) axis and the autonomic nervous system in IBS, have attracted increasing attention. The objective of this study was to examine the impact of paeoniflorin (PF) on anxiety and visceral hypersensitivity in maternal separation-induced IBS-like rats. The IBS-like rat model was established through the implementation of Maternal Separation (MS) and subsequently subjected to various doses of PF administered via oral gavage for 14 days. Anxiety-like behavior was evaluated using the Open Field Test (OFT) and Elevated Plus Maze (EPM) test. The assessment of visceral sensitivity involved the utilization of the Abdominal Withdrawal Reflex (AWR) score and electromyographic (EMG) responses of the external oblique muscle in response to colorectal distention. The levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), and corticotrophin-releasing hormone (CRH) were examined by ELISA. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were employed to detect the expressions of CRH receptors 1 (CRHR1) and 2 (CRHR2). Glucocorticoid receptors (GR), mineralocorticoid receptor (MR), brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB), and phospholipase C γ1 (PLCγ1) were examined by Western blot. The results showed that MS induced anxiety-like behavior and visceral hypersensitivity, while PF treatment attenuated these changes. Furthermore, the HPA axis hyperactivity in MS rats was attenuated by PF treatment, indicated by reduced serum ACTH, CORT, and CRH levels and recovered hippocampal CRHR1 and GR expressions. In addition, PF inhibited BDNF/TrkB signaling by downregulating the protein levels of BDNF, TrkB, and phospho-PLCγ1 in the colon. These findings suggest that PF alleviated anxiety and visceral hypersensitivity in MS-induced IBS-like rats, which may be the modulation of HPA axis activity and BDNF/TrkB/PLCγ1 signaling pathway.

Long-lasting adverse effects of short-term stress during the suckling–mastication transition period on masticatory function and intraoral sensation in rats

Early-life stress affects brain development, eventually resulting in adverse behavioral and physical health consequences in adulthood. The present study assessed the hypothesis that short-term early-life stress during infancy before weaning, a period for the maturation of mastication and sleep, poses long-lasting adverse effects on masticatory function and intraoral sensations later in life.Rat pups were exposed to either maternal separation (MS) or intermittent hypoxia (IH-Infancy) for 6 h/day in the light/sleep phase from postnatal day (P)17 to P20 to generate “neglect” and “pediatric obstructive sleep apnea” models, respectively. The remaining rats were exposed to IH during P45–P48 (IH-Adult). Masticatory ability was evaluated based on the rats’ ability to chew pellets and bite pasta throughout the growth period (P21–P70). Intraoral chemical and mechanical sensitivities were assessed using two-bottle preference drinking tests, and hind paw pain thresholds were measured in adulthood (after P60).No differences were found in body weight, grip force, and hind paw sensitivity in MS, IH-Infancy, and IH-Adult rats compared with naïve rats. Masticatory ability was lower in MS and IH-Infancy rats from P28 to P70 than in naïve rats. MS and IH-Infancy rats exhibited intraoral hypersensitivity to capsaicin and mechanical stimulations in adulthood. The IH-Adult rats did not display inferior masticatory ability or intraoral hypersensitivity.In conclusion, short-term early-life stress during the suckling–mastication transition period potentially causes a persistent decrease in masticatory ability and intraoral hypersensitivity in adulthood. The period is a “critical window” for the maturation of oral motor and sensory functions.

Involvement of oxidative stress in orofacial mechanical pain hypersensitivity following neonatal maternal separation in rats

Patients with persistent pain have sometimes history of physical abuse or neglect during infancy. However, the pathogenic mechanisms underlying orofacial pain hypersensitivity associated with early-life stress remain unclear. The present study focused on oxidative stress and investigated its role in pain hypersensitivity in adulthood following early-life stress. To establish an early-life stress model, neonatal pups were separated with their mother in isolated cages for 2 weeks. The mechanical head-withdrawal threshold (MHWT) in the whisker pad skin of rats received maternal separation (MS) was lower than that of non-MS rats at postnatal week 7. In MS rats, the expression of 8-hydroxy-deoxyguanosine, a marker of DNA oxidative damage, was enhanced, and plasma antioxidant capacity, but not mitochondrial complex I activity, decreased compared with that in non-MS rats. Reactive oxygen species (ROS) inactivation and ROS-sensitive transient receptor potential ankyrin 1 (TRPA1) antagonism in the whisker pad skin at week 7 suppressed the decrease of MHWT. Corticosterone levels on day 14 increased in MS rats. Corticosterone receptor antagonism during MS periods suppressed the reduction in antioxidant capacity and MHWT. The findings suggest that early-life stress potentially induces orofacial mechanical pain hypersensitivity via peripheral nociceptor TRPA1 hyperactivation induced by oxidative stress in the orofacial region.

The role of SIRT3 in mediating the cognitive deficits and neuroinflammatory changes associated with a developmental animal model of schizophrenia

The neuroinflammatory state may contribute to the pathogenesis of many mental disorders including schizophrenia. Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for activation of proteins involved in mitochondria quality control, such as Sirtuin3 (SIRT3). Our previous study has found that NAD+ supplement could rescue early life stress (ELS)-induced neuroinflammation and down-regulation of SIRT3 in adult offspring. However, it is unclear whether SIRT3 is the key to the neuroprotective effects of NAD+ supplement in this animal model of schizophrenia. The present study used 24 h maternal separation (MS) as ELS to Wistar rat pups on the postnatal day (PND) 9. Schizophrenia-like behaviors and memory impairments were detected by behavioral tests. Microglial activation, pro-inflammatory cytokine expression, and NAD+/SIRT3 expression were detected in the prefrontal cortex and hippocampus. Meanwhile, NAM (a precursor of NAD+), and the SIRT3 activator Honokiol (HNK), and the SIRT3 inhibitor 3-TYP were used as an intervention in vivo. Our results showed that ELS could induce schizophrenia-like behaviors and M1 microglial activation, NAD+ decline, lower expression of SIRT3, and increased acetylated superoxide dismutase 2 expression at the adult stage. NAD+ supplement or HNK administration could block this process and normalize the behavioral alterations of the MS animals. 3-TYP administration in the control group and the NAM-treated MS rats caused M1 microglial activation and cognitive deficits. Our results demonstrated that SIRT3 mediated the stabilizing effect of NAD+ on normalizing M1 microglial activation and behavioral phenotypes in MS rats.

Capsaicin alleviates neuronal apoptosis and schizophrenia-like behavioral abnormalities induced by early life stress

Early life stress (ELS) is associated with the later development of schizophrenia. In the rodent model, the maternal separation (MS) stress may induce neuronal apoptosis and schizophrenia-like behavior. Although the TRPV1 agonist capsaicin (CAP) has been reported to reduce apoptosis in the central nervous system, its effect in MS models is unclear. Twenty-four hours of MS of Wistar rat pups on postnatal day (PND9) was used as an ELS. Male rats in the adult stage were the subjects of the study. CAP (1 mg/kg/day) intraperitoneal injection pretreatment was undertaken before behavioral tests for 1 week and continued during the tests. Behavioral tests included open field, novel object recognition, Barnes maze test, and pre-pulse inhibition (PPI) test. MS rats showed behavioral deficits and cognitive impairments mimicking symptoms of schizophrenia compared with controls. MS decreased the expression of TRPV1 in the frontal association cortex (FrA) and in the hippocampal CA1, CA3, and dentate gyrus (DG) regions compared with the control group resulting in the increase of pro-apoptotic proteins (BAX, Caspase3, Cleaved-Caspase3) and the decrease of anti-apoptotic proteins (Bcl-2). The number of NeuN++TUNEL+ cells increased in the MS group in the FrA, CA1, CA3, and DG compared with the control group. Neuronal and behavioral impairments of MS were reversed by treatment with CAP. Exposure to ELS may lead to increased neuronal apoptosis and impaired cognitive function with decreased TRPV1 expression in the prefrontal cortex and hippocampus in adulthood. Sustained low-dose administration of CAP improved neuronal apoptosis and cognitive function. Our results provide evidence for future clinical trials of chili peppers or CAP as dietary supplements for the reversal treatment of schizophrenia.

Gut instinct: Sex differences in the gut microbiome are associated with changes in adolescent nociception following maternal separation in rats.

Adolescent chronic pain is a growing public health epidemic. Our understanding of its etiology is limited; however, several factors can increase susceptibility, often developing in response to an acute pain trigger such as a surgical procedure or mild traumatic brain injury (mTBI), or an adverse childhood experience (ACE). Additionally, the prevalence and manifestation of chronic pain is sexually dimorphic, with double the rates in females than males. Despite this, the majority of pre-clinical pain research focuses on males, leaving a gap in mechanistic understanding for females. Given that emerging evidence has linked the gut microbiome and the brain-gut-immune axis to various pain disorders, we aimed to investigate sex-dependent changes in taxonomic and functional gut microbiome features following an ACE and acute injury as chronic pain triggers. Male and female Sprague Dawley rat pups were randomly assigned to either a maternal separation (MS) or no stress paradigm, then further into a sham, mTBI, or surgery condition. Chronically, the von Frey test was used to measure mechanical nociception, and fecal samples were collected for 16S rRNA sequencing. Animals in the surgery group had an increase in pain sensitivity when compared to mTBI and sham groups, and this was complemented by changes to the gut microbiome. In addition, significant sex differences were identified in gut microbiome composition, which were exacerbated in response to MS. Overall, we provide preliminary evidence for sex differences and ACE-induced changes in bacterial composition that, when combined, may be contributing to heterogeneity in pain outcomes.

Electroacupuncture-modulated extracellular ATP levels in prefrontal cortex ameliorated depressive-like behavior of maternal separation rats

Maternal separation (MS) is a type of early-life stress that has been linked to neuropsychiatric disorders, especially depression. Increasing evidence indicates that the adenosine triphosphate (ATP) level in the prefrontal cortex (PFC) is involved in the pathophysiology of depression. To investigate the potential relationship between ATP in PFC and antidepressant effects of electroacupuncture (EA) treatment, we assessed genes involved in ATP biosynthesis as well as the extracellular ATP levels in a rat model exposed to neonatal MS. Our results demonstrated that reduced expression of ABCG2 (an ATP-binding cassette protein) and ATP levels in the PFC of depressive-like rats exposed to MS can be attenuated by EA stimulus at the Baihui (GV20) and Yintang (GV29) acupoints. Moreover, the antidepressant effect of EA treatment was blocked by administration of suramin, a broad purinergic P2 receptor antagonist. Together, these results suggested that electroacupuncture may be able to modulate extracellular ATP levels in the PFC of depressive-like MS rats, potentially contributing to its antidepressant effects.

Nicotinamide ameliorates mitochondria-related neuronal apoptosis and cognitive impairment via the NAD+/SIRT3 pathway

Emerging evidence suggests that mitochondria play a central role in mental health disorders including schizophrenia. Here we investigated whether nicotinamide (NAM) normalized cognitive impairment via a mechanism involving the mitochondrial Sirtuin 3 (SIRT3) pathway. The 24 h maternal separation (MS) rat model was used to mimic schizophrenia-associate phenotypes. Schizophrenia-like behaviors and memory impairments were detected using the pre-pulse inhibition test, novel object recognition test, and Barnes maze test, and neuronal apoptosis was characterized using multiple assays. SIRT3 activity was inhibited pharmacologically or by knockdown in HT22 cells, and BV2 microglia and SIRT3-knockdown HT22 cells were co-cultured in vitro. Mitochondrial molecules were measured by western blotting, and mitochondrial damage was measured with reactive oxygen species and mitochondrial membrane potential assays. Proinflammatory cytokines were assayed by ELISA and microglial activation was detected by immunofluorescence. MS animals showed behavioral and cognitive impairment and increased neuronal apoptosis. Supplementation with NAM or administration of honokiol, a SIRT3 activator, reversed all of the changes in behavioral and neuronal phenotypes. Administration of the SIRT3 inhibitor 3-TYP in control and NAM-treated MS rats caused behavioral and neuronal phenotypes similar to MS. In vitro, inhibition of SIRT3 activity with 3-TYP or by knockdown in HT22 cells increased ROS accumulation and caused neuronal apoptosis in a single-culture system. In co-culture systems, SIRT3 knockdown in HT22 cells activated BV2 microglia and increased levels of TNF-α, IL-6, and IL-1β. The administration of NAM blocked these alterations. Taken together, these data suggest that NAM can rescue neuronal apoptosis and microglial over-activation through the nicotinamide adenine dinucleotide (NAD+)-SIRT3-SOD2 signaling pathway, furthering our understanding of the pathogenesis of schizophrenia and providing avenues for novel treatments.

Protective effects of vanillic acid on autistic-like behaviors in a rat model of maternal separation stress: Behavioral, electrophysiological, molecular and histopathological alterations.

Compounds derived from herbs exhibit a range of biological properties, including anti-inflammatory, antioxidant, and neuroprotective properties. However, the exact mechanism of action of these compounds in various neurological disorders is not fully discovered yet. Herein, the present work detected the effect of Vanillic acid (VA), a widely-used flavoring agent derived from vanillin, on autistic-like behaviors to assess the probable underlying mechanisms that mediate behavioral, electrophysiological, molecular, and histopathological alterations in the rat model of maternal separation (MS) stress. Maternal separated rats were treated with VA (25, 50, and 100mg/kg interperitoneally for 14days). In addition, anxiety-like, autistic-like behaviors, and learning and memory impairment were evaluated using various behavioral tests. Hippocampus samples were assessed histopathologically by H&E staining. Levels of malondialdehyde (MDA) and antioxidant capacity (by the FRAP method), as well as nitrite levels, were measured in brain tissue. Moreover, gene expression of inflammatory markers (IL-1β, TLR-4, TNF-α, and NLRP3) was evaluated in the hippocampus. Electrophysiological alterations were also estimated in the hippocampus by long-term potentiation (LTP) assessments. Results showed that VA reversed the negative effects of MS on behavior. VA increased the diameter and decreased the percentage of dark neurons in the CA3 area. Accordingly, VA decreased MDA and nitrite levels and increased the antioxidant capacity in brain samples and decreased the expression of all inflammatory genes. VA treated rats showed significant improvements in all LTP parameters. This study provided evidence suggesting a possible role for VA in preventing autism spectrum disorder (ASD) by regulating immune signaling.

Chronic morphine exposure and maternal separation induce impairments in cognition and locomotor activity of male adolescent rats.

Maternal morphine exposure reduces motivation for basic cognitive tasks, followed by executive function deficits in attention and accuracy. It also induces depression-like behaviors and has negative consequences for learning and memory in offspring. Interaction between mothers and pups has a crucial effect on the mammal's development. Maternal separation (MS) can originate behavioral and neuropsychiatric abnormalities later in life. It seems that adolescents are more susceptible to the effects of early-life stress; Therefore, this study aimed to evaluate the effects of chronic morphine consumption (21 days before and after mating and gestation) and MS (180 min/day from postnatal day [PND] 1-21) on the cognitive and behavioral performance of male offspring in mid-adolescence. Six groups, including control, MS, V (vehicle), morphine, V+MS, and morphine+MS, were tested for open field (OF), novel object recognition (NOR), and the Morris water maze (MWM). The results of the OF test showed that MS increased locomotor activity and movement velocity. Inner and outer zone durations did not differ among groups. The body stretching of the morphine+MS rats was significantly more than the MS rats. Moreover, the MS and morphine+MS groups showed significantly less sniffing behavior in the OF test. The MS group showed deficits in spatial learning in the MWM test, but recognition memory in the NOR and spatial memory in the MWM tests were not significantly different among groups. We concluded that MS could induce impairments in spatial learning and locomotor activity that could be worsened by maternal morphine exposure in adolescent male rats.

Modulation of the endoplasmic reticulum stress and unfolded protein response mitigates the behavioral effects of early-life stress

BackgroundEarly-life stress (ELS) affects brain development and increases the risk of mental disorders associated with the dysfunction of the medial prefrontal cortex (mPFC). The mechanisms of ELS action are not well understood. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are cellular processes involved in brain maturation through the regulation of pro-survival or proapoptotic processes. We hypothesized that ER stress and the UPR in the mPFC are involved in the neurobiology of ELS.MethodsWe performed a maternal separation (MS) procedure from postnatal days 1 to 14 in rats. Before each MS, pups were injected with an inhibitor of ER stress, salubrinal or a vehicle. The mRNA and protein expression of UPR and apoptotic markers were evaluated in the mPFC using RT-qPCR and Western blot methods, respectively. We also estimated the numbers of neurons and glial cells using stereological methods. Additionally, we assessed behavioral phenotypes related to fear, anhedonia and response to psychostimulants.ResultsMS slightly enhanced the activation of the UPR in juveniles and modulated the expression of apoptotic markers in juveniles and preadolescents but not in adults. Additionally, MS did not affect the numbers of neurons and glial cells at any age. Both salubrinal and vehicle blunted the expression of UPR markers in juvenile and preadolescent MS rats, often in a treatment-specific manner. Moreover, salubrinal and vehicle generally alleviated the behavioral effects of MS in preadolescent and adult rats.ConclusionsModulation of ER stress and UPR processes may potentially underlie susceptibility or resilience to ELS.

Preventive effect of quercetin-Loaded nanophytosome against autistic-like damage in maternal separation model: The possible role of Caspase-3, Bax/Bcl-2 and Nrf2

The autism is an abnormality in the neuronal advance which starts before age 3 recognized by defective behaviors. This study aimed to make quercetin-loaded nanophytosomes (QNP) on behavioral deficits, cerebellar oxidative stress and apoptosis in an autistic-like model caused by maternal separation (MS). The newborn rats are randomly categorized into seven groups, including control, positive control, disease, and diseases treated with quercetin (10 and 40 mg/kg) and QNP (10 and 40 mg/kg). Pups exposed to MS for 3 h per day from postnatal days (PND) 1–9 showed behavioral impairment in adult rats compared to control group. The oral administration of quercetin and QNP was constantly started after the lactation period (21 postnatal days) for three weeks. Autistic-like behaviors, antioxidant parameters, and Nrf2, Bax/Bcl-2, and Caspase-3 expressions were surveyed in the cerebellum. Quercetin (40 mg/kg) treated improved some behavioral disorders. Also, the improvement of oxidative stress parameters, Nrf2 and apoptotic factors gene expression was observed in the cerebellum of quercetin (40 mg/kg) treated (p < 0.01). QNP treatment (10 and 40 mg/kg) significantly ameliorated anxiety-like behaviors, line crossing, and grooming index (p < 0.001), lipid peroxidation (p < 0.001), and increased catalase (CAT) (p < 0.001), superoxide dismutase (SOD) (p < 0.001), glutathione peroxidase (GPx) (p < 0.001) activity, and glutathione (GSH) levels (p < 0.05). Moreover, QNP significantly reduced Caspase-3 and Bax expression (p < 0.001), but increased Bcl-2, and Nrf2 expressions (p < 0.001). These findings indicated that QNP due to its high bioavailability was more effective than quercetin can be reduced autistic-like behavior, oxidative and apoptotic damages in the model of MS rats.

Aqueous cinnamon extract ameliorates bowel dysfunction and enteric 5-HT synthesis in IBS rats.

Cinnamon protects against irritable bowel syndrome with diarrhea (IBS-D) in humans, but its efficacy and underlying mechanism of action remain poorly understood. Maternally separated (MS) IBS-D rat model and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced post-inflammatory IBS-D rat model are characterized by visceral hyperalgesia and diarrhea. This study used the two models to evaluate the effect of cinnamon extract (CE) on bowel symptoms. The MS rat model was also used to explore its underlying anti-IBS mechanism. cinnamon extract reduced defecation frequency and visceral hyperalgesia in MS rats in a dose-dependent manner and effectively improved visceral hyperalgesia in TNBS rats. The efficacy of cinnamon extract was comparable to the positive drug serotonin receptor 3 (5-HT3) selective antagonist, Ramosetron. Excessive 5-HT, a well-known pathogenic factor for IBS, in the colon and circulation of IBS rats was reduced after cinnamon extract intervention. Both, gene and protein levels of the colonic 5-HT synthetase, Tryptophan Hydroxylase 1 (Tph1), were also decreased in CE-treated IBS rats. In addition, a luciferase assay revealed that cinnamon extract and its major components, catechin, procyanidin B1/2, cinnamic acid, and cinnamyl alcohol, significantly inhibited Tph1 transcription activity in vitro. These findings illustrated that aqueous cinnamon extract partially attenuated bowel symptoms in IBS models by directly inhibiting Tph1 expression and controlling 5-HT synthesis. This provides a scientific viewpoint for the use of cinnamon as a folk medication to treat IBS.

Leucine mediates cognitive dysfunction in early life stress-induced mental disorders by activating autophagy.

To explore the relationship between leucine in cerebrospinal fluid (CSF) and cognitive dysfunction in rats with early life stress (ELS) induced mental illness, and pathophysiological mechanism involved. The maternal separation (MS), an animal paradigm used widely as a preclinical model of ELS which is one of the important risk factors for mental disorders. Behavioral experiments including open-field test, sucrose preference, object recognition and Morris water maze tests, Nissl staining, transmission electron microscopy and WES were employed in the present study. The behavioral results showed that MS rats were more prone to cognitive impairment and depression-and-anxiety-like behaviors than controls, including spatial self-exploration ability, memory ability, and spatial learning and memory function. Nissl staining analysis indicated that the number of neurons in the CA1 and CA3 regions of the hippocampus significantly decreased and the arrangement of nerve cells was abnormal. The leucine levels were decreased in the CSF of MS rats and highly correlated with the number of hippocampal neurons, and yet leucine supplementation improved the degree of MS-induced cognitive impairment. Furthermore, there were autophagosomes in the hippocampus of the low-leucine diet rats of the control and MS group but not in the high-leucine diet MS group by transmission electron microscopy. The protein expression of Beclin-1 in the hippocampus was significantly increased in the MS normal diet group and MS low-leucine diet group, yet decreased in the MS high-leucine diet group compared with the MS low-leucine diet group. Meanwhile, the Bcl-2/Bax ratio was significantly decreased in the control low-leucine diet group, MS normal diet group and MS low-leucine diet group. Ultimately, in vitro experiments suggested that leucine deficiency could activate neuronal autophagy including enhanced LC3II/LC3I and mRFP-GFP-LC3, which was consistent with the in vivo results, and the cell apoptosis rate and lactate dehydrogenase (LDH) cytotoxicity were also increased with leucine deficiency, while the above effects could be partly reversed by autophagy inhibitor treatment. MS model caused adult male rats to be susceptible to cognitive dysfunction, which may regulate autophagy in hippocampal neurons through leucine metabolism in CSF.

Aversive memory reactivation: A possible role for delta oscillations in the hippocampus-amygdala circuit.

Memory labilization, the process by which memories become susceptible to update, is essential for memory reconsolidation and has been a target for novel therapies for traumatic memory-associated disorders. Maternal separation (MS) in male rats produced memories resistant to labilization in adulthood. Based on previous results, we hypothesized that temporal desynchronization between the dorsal hippocampus (DHc) and the basolateral amygdala (BLA), during memory retrieval, could be responsible for this impairment. Our goal was to investigate possible differences in oscillatory activity and synchrony between the DHc and BLA during fear memory reactivation, between MS and non-handled (NH) rats. We used male adult Wistar rats, NH or MS, with electrodes for local field potential (LFP) recordings implanted in the DHc and BLA. Animals were submitted to aversive memory reactivation by exposure to the conditioned context (Reat) or to pseudo-reactivation in a neutral context (pReat), and LFP was recorded. Plasticity markers linked to reconsolidation were evaluated one hour after reactivation. The power of delta oscillations and DHc-BLA synchrony in Reat animals was increased, during freezing. Besides, delta modulation of gamma oscillations amplitude in the BLA was associated with the increase in DHc Zif268 levels, an immediate early gene specifically associated with reconsolidation. Concerning early life stress, we found lower power of delta and strength of delta-gamma oscillations coupling in MS rats, compared to NH, which could explain the low Zif268 levels in a subgroup of MS animals. These results suggest a role for delta oscillations in memory reactivation that should be further investigated.

Sinisan alleviates depression-like behaviors by regulating mitochondrial function and synaptic plasticity in maternal separation rats

BackgroundSinisan (SNS) consists of four kinds of herbs, which is the core of antidepressant prescription widely used in traditional Chinese medicine clinic treatment for depression induced by early life stress. However, the role and precise mechanism of SNS antidepressant have not yet been elucidated. PurposeThis study aimed to investigate the mechanism SNS on antidepressant of regulating mitochondrial function to improve hippocampal synaptic plasticity. Methods90 Sprague-Dawley (SD) rats male pups on Post-Natal Day (PND) 0 were randomly divided into Control group (ddH20), Model group (ddH20), Fluoxetine group (5.0 mg/kg fluoxetine), and SNS-L group (2.5 g/kg SNS), SNS-M group (5.0 g/kg SNS) and SNS-H group (10.0 g/kg SNS), 15 animals per group. Maternal separation (MS) from PND1 to PND21, drug intervention from PND60 to PND90, and behavior tests including sucrose preference test, open field test and forced swimming test from PND83 to PND90 were performed. Synaptic structure and mitochondrial structure were observed by TEM. The expression levels of PSD-95 and SYN were detected by immunohistochemistry and western blot test, the adenosine triphosphate (ATP) content in the hippocampus was detected by assay kits, and the expression levels of Mfn2, Drp1 and Fis1 protein were detected by western bolt test. ResultsSNS can alleviate depression-like and anxiety-like behaviors in MS rats, improve the damage of synapses and mitochondria, reduce the decrease of ATP in hippocampus, and reverse the expression levels of PSD-95, SYN, Mfn2, Drp1, and Fis1 proteins. ConclusionSNS reduced the risk of early life stress induced depression disorder via regulating mitochondrial function and synaptic plasticity. Targeting mitochondrial may be a novel prospective therapeutic avenue for antidepressant.

Upregulation of Netrin-1 in the hippocampus mediates the formation of visceral hypersensitivity induced by maternal separation

Early adverse life events (EALs), such as maternal separation (MS), can cause visceral hypersensitivity, which is thought to be a key pathophysiological mechanism of irritable bowel syndrome (IBS). Previous studies mainly focused on EALs-induced visceral hypersensitivity in adulthood but did not consider that it may have occurred in the preadult period. We previously found that rats who experienced MS suffered from visceral hypersensitivity starting from the post-weaning period. Moreover, the hippocampus is considered to be critical in regulating the formation of visceral hypersensitivity induced by MS. But the underlying mechanisms throughout different life periods are unclear. In this study, behavioral tests, RNA-seq, lentiviral interference, and molecular biology techniques were applied to investigate the molecular mechanism in the hippocampus underlying MS-induced long-lasting visceral hypersensitivity. It was found that both visceral sensitivity and anxiety-like behaviors were significantly increased in MS rats in post-weaning, prepubertal, and adult periods, especially in the prepubertal period. Subsequently, RNA-seq targeting the hippocampus identified that the expression level of Netrin-1 was significantly increased in all periods, which was further confirmed by quantitative real-time PCR and Western blot. Knocking-down hippocampal Netrin-1 in the post-weaning period by lentivirus interference alleviated visceral hypersensitivity and anxiety-like behaviors of MS rats in the later phase of life. In addition, deleted in colorectal cancer (DCC), instead of neogenin-1(Neo-1) or uncoordinated (UNC5), was proved to be the specific functional receptor of Netrin-1 in regulating visceral hypersensitivity, whose upregulation may result in the most severe symptoms in the prepubertal period. Furthermore, the activation of the Netrin-1/DCC pathway could enhance long-term potentiation (LTP) in the hippocampus, probably via recruitment of the AMPA receptor subunit GluA1, which finally resulted in the formation of visceral hypersensitivity. These novel findings suggest that long-lasting over-expression of Netrin-1 can mediate visceral hypersensitivity and anxiety disorder from the post-weaning period to adulthood by activating DCC/GluA1 pathway in the hippocampus. Moreover, early intervention of Netrin-1 in the post-weaning period could lead to significant symptom relief afterward, which provides evidence that the Netrin-1/DCC/GluA1 signaling pathway may be a potential therapeutic target for the treatment of visceral hypersensitivity in clinics.