Strain-specific changes in nucleus accumbens transcriptome and motivation for palatable food reward in mice exposed to maternal separation.

Introduction: In humans, adversity in childhood exerts enduring effects on brain and increases the vulnerability to psychiatric diseases. It also leads to a higher risk of eating disorders and obesity. Maternal separation (MS) in mice has been used as a proxy of stress during infancy. We hypothesized that MS in mice affects motivation to obtain palatable food in adulthood and changes gene expression in reward system. Methods: Male and female pups from C57Bl/6J and C3H/HeN mice strains were subjected to a daily MS protocol from postnatal day (PND) 2 to PND14. At adulthood, their motivation for palatable food reward was assessed in operant cages. Results: Compared to control mice, male and female C3H/HeN mice exposed to MS increased their instrumental response for palatable food, especially when the effort required to obtain the reward was high. Importantly, this effect is shown in animals fed ad libitum. Transcriptional analysis revealed 375 genes differentially expressed in the nucleus accumbens of male MS C3H/HeN mice compared to the control group, some of these being associated with the regulation of the reward system (e.g., Gnas, Pnoc). Interestingly, C57Bl/6J mice exposed to MS did not show alterations in their motivation to obtain a palatable reward, nor significant changes in gene expression in the nucleus accumbens. Conclusion: MS produces long-lasting changes in motivation for palatable food in C3H/HeN mice, but has no impact in C57Bl/6J mice. These behavioral alterations are accompanied by drastic changes in gene expression in the nucleus accumbens, a key structure in the regulation of motivational processes.

Impact of dietary vitamin A on striatal function in adult rats.

The striatum is a brain structure involved in the control of voluntary movement. Striatum contains high amounts of retinoic acid, the active metabolite of vitamin A, as well as retinoid receptors, RARß and RXRγ. Previous studies revealed that disruption of retinoid signaling initiated during development is deleterious for striatal physiology and related motor functions. However, the alteration of retinoid signaling, and the importance of vitamin A supply during adulthood on striatal physiology and function has never been established. In the present study, we investigated the impact of vitamin A supply on striatal function. Adult Sprague-Dawley rats were fed with three specific diets, either sub-deficient, sufficient, or enriched in vitamin A (0.4, 5, and 20 international units [IU] of retinol per g of diet, respectively) for 6 months. We first validated that vitamin A sub-deficient diet in adult rats constitutes a physiological model of retinoid signaling reduction in the striatum. We then revealed subtle alterations of fine motor skills in sub-deficient rats using a new behavioral apparatus specifically designed to test forepaw reach-and-grasp skills relying on striatal function. Finally, we showed using qPCR analysis and immunofluorescence that the striatal dopaminergic system per se was not affected by vitamin A sub-deficiency at adult age. Rather, cholinergic synthesis in the striatum and µ-opioid receptor expression in striosomes sub-territories were the most affected by vitamin A sub-deficiency starting at adulthood. Taken together these results revealed that retinoid signaling alteration at adulthood is associated with motor learning deficits together with discrete neurobiological alterations in the striatum.

Microbiota and stress: a loop that impacts memory.

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower α and ß diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

A new experimental design to study inflammation-related versus non-inflammation-related depression in mice.

BACKGROUND: Major depressive disorder (MDD) represents a major public health concern, particularly due to its steadily rising prevalence and the poor responsiveness to standard antidepressants notably in patients afflicted with chronic inflammatory conditions, such as obesity. This highlights the need to improve current therapeutic strategies, including by targeting inflammation based on its role in the pathophysiology and treatment responsiveness of MDD. Nevertheless, dissecting the relative contribution of inflammation in the development and treatment of MDD remains a major issue, further complicated by the lack of preclinical depression models suitable to experimentally dissociate inflammation-related vs. inflammation-unrelated depression. METHODS: While current models usually focus on one particular MDD risk factor, we compared in male C57BL/6J mice the behavioral, inflammatory and neurobiological impact of chronic exposure to high-fat diet (HFD), a procedure known to induce inflammation-related depressive-like behaviors, and unpredictable chronic mild stress (UCMS), a stress-induced depression model notably renowned for its responsivity to antidepressants. RESULTS: While both paradigms induced neurovegetative, depressive-like and anxiety-like behaviors, inflammation and downstream neurobiological pathways contributing to inflammation-driven depression were specifically activated in HFD mice, as revealed by increased circulating levels of inflammatory factors, as well as brain expression of microglial activation markers and enzymes from the kynurenine and tetrahydrobiopterin (BH4) pathways. In addition, serotoninergic and dopaminergic systems were differentially impacted, depending on the experimental condition. CONCLUSIONS: These data validate an experimental design suitable to deeply study the mechanisms underlying inflammation-driven depression comparatively to non-inflammatory depression. This design could help to better understand the pathophysiology of treatment resistant depression.

Transcriptomic signaling pathways involved in a naturalistic model of inflammation-related depression and its remission.

This study aimed at identifying molecular biomarkers of inflammation-related depression in order to improve diagnosis and treatment. For this, we performed whole-genome expression profiling from peripheral blood in a naturalistic model of inflammation-associated major depressive disorder (MDD) represented by comorbid depression in obese patients. We took advantage of the marked reduction of depressive symptoms and inflammation following bariatric surgery to test the robustness of the identified biomarkers. Depression was assessed during a clinical interview using Mini-International Neuropsychiatric Interview and the 10-item, clinician-administered, Montgomery-Asberg Depression Rating Scale. From a cohort of 100 massively obese patients, we selected 33 of them for transcriptomic analysis. Twenty-four of them were again analyzed 4-12 months after bariatric surgery. We conducted differential gene expression analyses before and after surgery in unmedicated MDD and non-depressed obese subjects. We found that TP53 (Tumor Protein 53), GR (Glucocorticoid Receptor), and NFκB (Nuclear Factor kappa B) pathways were the most discriminating pathways associated with inflammation-related MDD. These signaling pathways were processed in composite z-scores of gene expression that were used as biomarkers in regression analyses. Results showed that these transcriptomic biomarkers highly predicted depressive symptom intensity at baseline and their remission after bariatric surgery. While inflammation was present in all patients, GR signaling over-activation was found only in depressed ones where it may further increase inflammatory and apoptosis pathways. In conclusion, using an original model of inflammation-related depression and its remission without antidepressants, we provide molecular predictors of inflammation-related MDD and new insights in the molecular pathways involved.

Gut microbiota resilience in horse athletes following holidays out to pasture.

Elite horse athletes that live in individual boxes and train and compete for hours experience long-term physical and mental stress that compromises animal welfare and alters the gut microbiota. We therefore assessed if a temporary period out to pasture with conspecifics could improve animal welfare and in turn, favorably affect intestinal microbiota composition. A total of 27 athletes were monitored before and after a period of 1.5 months out to pasture, and their fecal microbiota and behavior profiles were compared to those of 18 horses kept in individual boxes. The overall diversity and microbiota composition of pasture and control individuals were temporally similar, suggesting resilience to environmental challenges. However, pasture exposure induced an increase in Ruminococcus and Coprococcus that lasted 1-month after the return to individual boxes, which may have promoted beneficial effects on health and welfare. Associations between the gut microbiota composition and behavior indicating poor welfare were established. Furthermore, withdrawn behavior was associated with the relative abundances of Lachnospiraceae AC2044 group and Clostridiales family XIII. Both accommodate a large part of butyrate-producing bacterial genera. While we cannot infer causality within this study, arguably, these findings suggest that management practices maintained over a longer period of time may moderate the behavior link to the gut ecosystem beyond its resilience potential.

Dietary vitamin A supplementation prevents early obesogenic diet-induced microbiota, neuronal and cognitive alterations.

BACKGROUND: Early consumption of obesogenic diets, rich in saturated fat and added sugar, is associated with a plethora of biological dysfunctions, at both peripheral and brain levels. Obesity is also linked to decreased vitamin A bioavailability, an essential molecule for brain plasticity and memory function. METHODS: Here we investigated in mice whether dietary vitamin A supplementation (VAS) could prevent some of the metabolic, microbiota, neuronal and cognitive alterations induced by obesogenic, high-fat and high-sugar diet (HFSD) exposure from weaning to adulthood, i.e. covering periadolescent period. RESULTS: As expected, VAS was effective in enhancing peripheral vitamin A levels as well as hippocampal retinoic acid levels, the active metabolite of vitamin A, regardless of the diet. VAS attenuated HFSD-induced excessive weight gain, without affecting metabolic changes, and prevented alterations of gut microbiota α-diversity. In HFSD-fed mice, VAS prevented recognition memory deficits but had no effect on aversive memory enhancement. Interestingly, VAS alleviated both HFSD-induced higher neuronal activation and lower glucocorticoid receptor phosphorylation in the hippocampus after training. CONCLUSION: Dietary VAS was protective against the deleterious effects of early obesogenic diet consumption on hippocampal function, possibly through modulation of the gut-brain axis.

Spontaneous intake of essential oils after a negative postnatal experience has long-term effects on blood transcriptome in chickens.

Chicks subjected to early stressful factors could develop long-lasting effects on their performances, welfare and health. Free access to essential oils (EO) in poultry farming could mitigate these effects and potentially reduce use of antimicrobial drugs. This study on chicken analyzed long-lasting effects of post-hatch adverse conditions (Delayed group), and the impact of EO intake on blood physiological parameters and transcriptome. Half of the Control and Delayed groups had free access to EO, while the other half had only water for the first 13 days post-hatching. Blood analyses of metabolites, inflammation and oxidative stress biomarkers, and mRNA expression showed sex differences. Long-lasting effects of postnatal experience and EO intake persisted in blood transcriptome at D34. The early adverse conditions modified 68 genes in males and 83 genes in females. In Delayed males six transcription factors were over-represented (NFE2L2, MEF2A, FOXI1, Foxd3, Sox2 and TEAD1). In females only one factor was over-represented (PLAG1) and four under-represented (NFIL3, Foxd3, ESR2 and TAL1::TCF3). The genes showing modified expression are involved in oxidative stress, growth, bone metabolism and reproduction. Remarkably, spontaneous EO intake restored the expression levels of some genes affected by the postnatal adverse conditions suggesting a mitigating effect of EO intake.

Priming for welfare: gut microbiota is associated with equitation conditions and behavior in horse athletes.

We simultaneously measured the fecal microbiota and multiple environmental and host-related variables in a cohort of 185 healthy horses reared in similar conditions during a period of eight months. The pattern of rare bacteria varied from host to host and was largely different between two time points. Among a suite of variables examined, equitation factors were highly associated with the gut microbiota variability, evoking a relationship between gut microbiota and high levels of physical and mental stressors. Behavioral indicators that pointed toward a compromised welfare state (e.g. stereotypies, hypervigilance and aggressiveness) were also associated with the gut microbiota, reinforcing the notion for the existence of the microbiota-gut-brain axis. These observations were consistent with the microbiability of behaviour traits (> 15%), illustrating the importance of gut microbial composition to animal behaviour. As more elite athletes suffer from stress, targeting the microbiota offers a new opportunity to investigate the bidirectional interactions within the brain gut microbiota axis.

Housing Horses in Individual Boxes Is a Challenge with Regard to Welfare.

Horses are mainly housed in individual boxes. This housing system is reported to be highly detrimental with regard to welfare and could trigger the expression of four behavioural indicators of a compromised welfare state: stereotypies, aggressiveness toward humans, unresponsiveness to the environment, and stress-related behaviours. The aim of this study was to identify housing and management factors that could alleviate the detrimental effects of individual boxes on welfare. A total of 187 horses were observed over 50 days by scan sampling. The impact of 12 factors was investigated on the expression of the four behavioural indicators in three different analyses. The results show that the majority of factors tested did not influence the expression of the behavioural indicators. Only three (straw bedding, a window opening onto the external environment, and a reduced quantity of concentrated feed) would have beneficial, although limited, effects. Furthermore, the longer the horses spent in individual boxes, the more likely they were to express unresponsiveness to the environment. To preserve the welfare of horses, it seems necessary to allow free exercise, interactions with conspecifics, and fibre consumption as often as possible, to ensure the satisfaction of the species' behavioural and physiological needs.

Progressive habituation to separation alleviates the negative effects of weaning in the mother and foal.

Early and definitive separation between offspring and their mothers has negative consequences on behavioral and physiological responses. This study compared sudden and definitive weaning (Sudd group, N = 16) and weaning involving progressive habituation to separation using a fence line during the month preceding definitive separation (Prog group, N = 18). The impact of these two methods was assessed in both foals and their mothers through behavioral and biological parameters, including salivary cortisol, telomere length and blood transcriptomes. On the day of definitive separation, Prog foals neighed and trotted less and presented lower cortisol levels than Sudd foals. The weaning type also acted on the foals' personality development; Prog foals became more curious, less fearful and less gregarious than Sudd foals, and the effects remained visible for at least 3 months. In principal component analysis, the Sudd and Prog groups were well separated along a factor where fear, reactivity and gregariousness correlated with high cortisol levels, but curiosity was associated with an increased telomere length and higher expression of genes involved in mitochondrial functions. Progressive weaning was also beneficial in mares. Principal component analysis showed that most Sudd group mares had higher cortisol levels and displayed more alert postures, neighs and activity on the day of weaning, indicating higher stress levels, while Prog mares had profiles that were characterized by more time spent resting on the day of weaning and longer telomere lengths. In conclusion, this study shows that progressive habituation to separation alleviates the negative effect of definitive weaning on both the mother and her young compared to sudden separation.

Opposite Expression of Hepatic and Pulmonary Corticosteroid-Binding Globulin in Cystic Fibrosis Patients.

Cystic fibrosis (CF) is characterized by a chronic pulmonary inflammation. In CF, glucocorticoids (GC) are widely used, but their efficacy and benefit/risk ratio are still debated. In plasma, corticosteroid-binding globulin (CBG) binds 90% of GC and delivers them to the inflammatory site. The main goal of this work was to study CBG expression in CF patients in order to determine whether CBG could be used to optimize GC treatment. The expression of CBG was measured in liver samples from CF cirrhotic and non-CF cirrhotic patients by qPCR and Western blot and in lung samples from non-CF and CF patients by qPCR. CBG binding assays with 3H-cortisol and the measurement of the elastase/α1-antitrypsin complex were performed using the plasmas. CBG expression increased in the liver at the transcript and protein level but not in the plasma of CF patients. This is possibly due to an increase of plasmatic elastase. We demonstrated that pulmonary CBG was expressed in the bronchi and bronchioles and its expression decreased in the CF lungs, at both levels studied. Despite the opposite expression of hepatic and pulmonary CBG in CF patients, the concentration of CBG in the plasma was normal. Thus, CBG might be useful to deliver an optimized synthetic GC displaying high affinity for CBG to the main inflammatory site in the context of CF, e.g., the lung.

The Effects of Weaning Methods on Gut Microbiota Composition and Horse Physiology.

Weaning has been described as one of the most stressful events in the life of horses. Given the importance of the interaction between the gut-brain axis and gut microbiota under stress, we evaluated (i) the effect of two different weaning methods on the composition of gut microbiota across time and (ii) how the shifts of gut microbiota composition after weaning affect the host. A total of 34 foals were randomly subjected to a progressive (P) or an abrupt (A) weaning method. In the P method, mares were separated from foals at progressively increasing intervals every day, starting from five min during the fourth week prior to weaning and ending with 6 h during the last week before weaning. In the A method, mares and foals were never separated prior to weaning (0 d). Different host phenotypes and gut microbiota composition were studied across 6 age strata (days -30, 0, 3, 5, 7, and 30 after weaning) by 16S rRNA gene sequencing. Results revealed that the beneficial species belonging to Prevotella, Paraprevotella, and Ruminococcus were more abundant in the A group prior to weaning compared to the P group, suggesting that the gut microbiota in the A cohort was better adapted to weaning. Streptococcus, on the other hand, showed the opposite pattern after weaning. Fungal loads, which are thought to increase the capacity for fermenting the complex polysaccharides from diet, were higher in P relative to A. Beyond the effects of weaning methods, maternal separation at weaning markedly shifted the composition of the gut microbiota in all foals, which fell into three distinct community types at 3 days post-weaning. Most genera in community type 2 (i.e., Eubacterium, Coprococcus, Clostridium XI, and Blautia spp.) were negatively correlated with salivary cortisol levels, but positively correlated with telomere length and N-butyrate production. Average daily gain was also greater in the foals harboring a community type 2 microbiota. Therefore, community type 2 is likely to confer better stress response adaptation following weaning. This study identified potential microbial biomarkers that could predict the likelihood for physiological adaptations to weaning in horses, although causality remains to be addressed.

Behavioral and Transcriptomic Fingerprints of an Enriched Environment in Horses (Equus caballus).

The use of environmental enrichment (EE) has grown in popularity over decades, particularly because EE is known to promote cognitive functions and well-being. Nonetheless, little is known about how EE may affect personality and gene expression. To address this question in a domestic animal, 10-month-old horses were maintained in a controlled environment or EE for 12 weeks. The control horses (n = 9) lived in individual stalls on wood shaving bedding. They were turned out to individual paddocks three times a week and were fed three times a day with pellets or hay. EE-treated horses (n = 10) were housed in large individual stalls on straw bedding 7 hours per day and spent the remainder of the time together at pasture. They were fed three times a day with flavored pellets, hay, or fruits and were exposed daily to various objects, odors, and music. The EE modified three dimensions of personality: fearfulness, reactivity to humans, and sensory sensitivity. Some of these changes persisted >3 months after treatment. These changes are suggestive of a more positive perception of the environment and a higher level of curiosity in EE-treated horses, explaining partly why these horses showed better learning performance in a Go/No-Go task. Reduced expression of stress indicators indicated that the EE also improved well-being. Finally, whole-blood transcriptomic analysis showed that in addition to an effect on the cortisol level, the EE induced the expression of genes involved in cell growth and proliferation, while the control treatment activated genes related to apoptosis. Changes in both behavior and gene expression may constitute a psychobiological signature of the effects of enrichment and result in improved well-being. This study illustrates how the environment interacts with genetic information in shaping the individual at both the behavioral and molecular levels.

Transcriptional responses of PBMC in psychosocially stressed animals indicate an alerting of the immune system in female but not in castrated male pigs.

BACKGROUND: Brain and immune system are linked in a bi-directional manner. To date, it remained largely unknown why immune components become suppressed, enhanced, or remain unaffected in relation to psychosocial stress. Therefore, we mixed unfamiliar pigs with different levels of aggressiveness. We separated castrated male and female pigs into psychosocially high- and low- stressed animals by skin lesions, plasma cortisol level, and creatine kinase activity obtained from agonistic behaviour associated with regrouping. Peripheral blood mononuclear cells (PBMC) were collected post-mortem and differential gene expression was assessed using the Affymetrix platform (n = 16). RESULTS: Relevant stress-dependent alterations were found only between female samples, but not between castrated male samples. Molecular routes related to TREM 1 signalling, dendritic cell maturation, IL-6 signalling, Toll-like receptor signalling, and IL-8 signalling were increased in high stressed females compared to low stressed females. This indicates a launch of immune effector molecules as a direct response. According to the shifts of transcripts encoding cell surface receptors (e.g. CD14, TLR2, TLR4, TREM1) the study highlights processes acting on pattern recognition, inflammation, and cell-cell communication. CONCLUSIONS: The transcriptional response partly affected the degree of 'stress responsiveness', indicating that the high stressed females altered their signal transduction due to potential infections and injuries while fighting.

Inflammation early in life is a vulnerability factor for emotional behavior at adolescence and for lipopolysaccharide-induced spatial memory and neurogenesis alteration at adulthood.

BACKGROUND: The postnatal period is a critical time window during which inflammatory events have significant and enduring effects on the brain, and as a consequence, induce alterations of emotional behavior and/or cognition later in life. However, the long-term effect of neonatal inflammation on behavior during adolescence, a sensitive period for the development of neurodevelopmental psychiatric disorders, has been little studied. In this study, we examined whether an early-life inflammatory challenge could alter emotional behaviors and spatial memory at adolescence and adulthood and whether stress axis activity, inflammatory response and neurogenesis were affected. METHODS: Lipopolysaccharide (LPS, 100 µg/kg) was administered to mice on postnatal day (PND) 14 and cytokine expression was measured in the plasma and in brain structures 3 hours later. Anxiety-like and depressive-like behavior (measured in the novelty-suppressed feeding test and the forced swim test, respectively) and spatial memory (Y-maze test) were measured at adolescence (PND30) and adulthood (PND90). Hypothalamic-pituitary-adrenal (HPA) axis activity (plasma corticosterone and glucocorticoid receptors in the hippocampus and prefrontal cortex) was measured at adulthood. In addition, the impact of a novel adult LPS challenge (100 µ/kg) was measured on spatial memory (Y-maze test), neurogenesis (doublecortin-positive cell numbers in the hippocampus) and plasma cytokine expression. RESULTS: First, we show in PND14 pups that a peripheral administration of LPS induced the expression of pro- and anti-inflammatory cytokines in the plasma and brain structures that were studied 3 hours after administration. Anxiety-like behavior was altered in adolescent, but not in adult, mice, whereas depressive-like behavior was spared at adolescence and increased at adulthood. This was accompanied by a decreased phosphorylation of the glucocorticoid receptor in the prefrontal cortex, with no effect on corticosterone levels. Second, neonatal LPS treatment had no effect on spatial memory in adolescence and adulthood. However, a second challenge of LPS in adulthood impaired spatial memory performance and neurogenesis and increased circulating levels of CCL2. CONCLUSIONS: Our study shows for the first time, in mice, that a peripheral LPS treatment at PND14 differentially alters emotional behaviors, but not spatial memory, at adolescence and adulthood. The behavioral effect of LPS at PND14 could be attributed to HPA axis deregulation and neurogenesis impairment.

Critical role of plasma corticosteroid-binding-globulin during stress to promote glucocorticoid delivery to the brain: impact on memory retrieval.

We aimed at demonstrating that corticosteroid binding globulin (CBG), a plasma glycoprotein binding glucocorticoids with high affinity in blood, endorses a major role under stress conditions by regulating free glucocorticoid access to the brain and thereby influences glucocorticoid-dependent behaviors. Hence, we compared CBG-deficient mice (Cbg-/-) and their controls (Cbg+/+) in a specific memory task, i.e. the delayed alternation behavior, requiring memory retrieval both under stress and nonstress conditions and previously shown to be dependent on hippocampal glucocorticoid levels. Our results evidence that Cbg-/- mice, unlike controls, remain insensitive to stress applied before memory retrieval. Furthermore, under stress conditions, we observed a blunted surge of corticosterone (CORT) in plasma and no free CORT rise in the hippocampus of Cbg-/-. Moreover, intrahippocampal infusion of CORT through implanted cannulae was used to mimic stress CORT rise before memory retrieval. This infusion of CORT reproduced memory retrieval impairments in Cbg-/- as in Cbg+/+ controls. Finally, we provide evidence that Cbg-/- mice exhibit a normal adrenal response to stress and ACTH. Given that CBG deficiency is known to markedly impact on CORT clearance from plasma, our current article demonstrates that Cbg-/- insensitivity in memory retrieval after stress results from the blunted CORT response due to increased CORT clearance. Overall, our data suggest that the impact of CBG genetic deficiency on various behavioral patterns reported previously stems from a smaller CORT reservoir in blood. Inasmuch as CBG discloses interindividual variations, such a parameter ought to be taken into account when studying stress-induced glucocorticoid action in brain.

Molecular genetics of hypothalamic-pituitary-adrenal axis activity and function.

The hypothalamic-pituitary-adrenocortical (HPA) axis is a major neuroendocrine system involved in the regulation of numerous physiological processes and in adaptation to stress. A wide range of variability can be observed in all the components of the system, and the contribution of genetic factors has been shown in the central regulation of the axis, the production of glucocorticoid hormones by the adrenal cortex, their bioavailability, and the efficiency of their action at the level of receptor and postreceptor mechanisms. Numerous molecular polymorphisms have been described that contribute to physiological variation as well as to HPA axis-related pathological conditions. Although most studies focus on single gene polymorphisms, future studies should aim to integrate the different sources of variation into a systems genetic model to take into account the strong interdependence of the different components of the axis.

Differential mRNA expression of genes in the porcine adrenal gland associated with psychosocial stress.

To gain insight into the adrenal stress response, we analysed differential mRNA expression of genes associated with psychosocial stress in the pig (Sus scrofa domestica). Various levels of psychosocial stress were induced by mixing groups of unfamiliar pigs with different aggressiveness. We selected two experimental groups for comparison, each comprising eight animals, which differed significantly in aggressive behaviour and plasma cortisol levels. To identify differentially expressed genes, we compared the adrenal transcriptome of these two groups of pigs, using the Affymetrix GeneChip porcine Genome Array. Bioinformatic analysis revealed that psychosocial stress induced upregulation of transcripts enriched for functions associated with cholesterol accumulation and downregulation of transcripts enriched for functions associated with cell growth and death. These responses are similar to those induced by ACTH stimulation. Nevertheless, the majority of the differentially expressed genes were so far not described as ACTH responsive. Some, such as GAL and GALP, may have responded to sympathoadrenal stimulation. Several of the differentially expressed transcripts, such as AGT, are associated with processes modulating steroidogenic response of adrenocortical cells to ACTH. One of the most significant findings was upregulation of LOC100039095, comprising a precursor of the microRNA miR-202, pointing to a previously unrecognised layer of regulation of adrenal steroidogenesis by microRNA. Our study, performed under entirely physiological conditions, complements previous studies focusing either on a single adrenal tissue and/or on a single stimulus, and contributes to understanding of the fine-tuning of adrenal stress response.

Association of HPA axis-related genetic variation with stress reactivity and aggressive behaviour in pigs.

BACKGROUND: Stress, elicited for example by aggressive interactions, has negative effects on various biological functions including immune defence, reproduction, growth, and, in livestock, on product quality. Stress response and aggressiveness are mutually interrelated and show large interindividual variation, partly attributable to genetic factors. In the pig little is known about the molecular-genetic background of the variation in stress responsiveness and aggressiveness. To identify candidate genes we analyzed association of DNA markers in each of ten genes (CRH g.233C>T, CRHR1 c.*866_867insA, CRHBP c.51G>A, POMC c.293_298del, MC2R c.306T>G, NR3C1 c.*2122A>G, AVP c.207A>G, AVPR1B c.1084A>G, UCN g.1329T>C, CRHR2 c.*13T>C) related to the hypothalamic-pituitary-adrenocortical (HPA) axis, one of the main stress-response systems, with various stress- and aggression-related parameters at slaughter. These parameters were: physiological measures of the stress response (plasma concentrations of cortisol, creatine kinase, glucose, and lactate), adrenal weight (which is a parameter reflecting activity of the central branch of the HPA axis over time) and aggressive behaviour (measured by means of lesion scoring) in the context of psychosocial stress of mixing individuals with different aggressive temperament. RESULTS: The SNP NR3C1 c.*2122A>G showed association with cortisol concentration (p = 0.024), adrenal weight (p = 0.003) and aggressive behaviour (front lesion score, p = 0.012; total lesion score p = 0.045). The SNP AVPR1B c.1084A>G showed a highly significant association with aggressive behaviour (middle lesion score, p = 0.007; total lesion score p = 0.003). The SNP UCN g.1329T>C showed association with adrenal weight (p = 0.019) and aggressive behaviour (front lesion score, p = 0.029). The SNP CRH g.233C>T showed a significant association with glucose concentration (p = 0.002), and the polymorphisms POMC c.293_298del and MC2R c.306T>G with adrenal weight (p = 0.027 and p < 0.0001 respectively). CONCLUSIONS: The multiple and consistent associations shown by SNP in NR3C1 and AVPR1B provide convincing evidence for genuine effects of their DNA sequence variation on stress responsiveness and aggressive behaviour. Identification of the causal functional molecular polymorphisms would not only provide markers useful for pig breeding but also insight into the molecular bases of the stress response and aggressive behaviour in general.