Effects of early-life stress on peripheral and central mitochondria in male mice across ages.

Exposure to early-life stress (ES) increases the vulnerability to develop metabolic diseases as well as cognitive dysfunction, but the specific biological underpinning of the ES-induced programming is unknown. Metabolic and cognitive disorders are often comorbid, suggesting possible converging underlying pathways. Mitochondrial dysfunction is implicated in both metabolic diseases and cognitive dysfunction and chronic stress impairs mitochondrial functioning. However, if and how mitochondria are impacted by ES and whether they are implicated in the ES-induced programming remains to be determined. ES was applied by providing mice with limited nesting and bedding material from postnatal day (P)2-P9, and metabolic parameters, cognitive functions and multiple aspects of mitochondria biology (i.e. mitochondrial electron transport chain (ETC) complex activity, mitochondrial DNA copy number, expression of genes relevant for mitochondrial function, and the antioxidant capacity) were studied in muscle, hypothalamus and hippocampus at P9 and late adulthood (10-12 months of age). We show that ES altered bodyweight (gain), adiposity and glucose levels at P9, but not in late adulthood. At this age, however, ES exposure led to cognitive impairments. ES affected peripheral and central mitochondria in an age-dependent manner. At P9, both muscle and hypothalamic ETC activity were affected by ES, while in hippocampus, ES altered the expression of genes involved in fission and antioxidant defence. In adulthood, alterations in ETC complex activity were observed in the hypothalamus specifically, whereas in muscle and hippocampus ES affected the expression of genes involved in mitophagy and fission, respectively. Our study demonstrates that ES affects peripheral and central mitochondria biology throughout life, thereby uncovering a converging mechanism that might contribute to the ES-induced vulnerability for both metabolic diseases and cognitive dysfunction, which could serve as a novel target for intervention.

Modulation of glucocorticoids by the serotonin transporter polymorphism: A narrative review.

The biological background and consequences of serotonin transporter polymorphism-glucocorticoid relationship in individual differences in stress reactivity has been a major interest in neuropsychiatry research. Individual differences in glucocorticoid release have long been implicated in vulnerability to stress-related psychopathologies, like depression and anxiety in various species. Yet, it is largely elusive to what extent results from non-human primates and rodents translate to human findings. Based on our structured, comprehensive and non-hypothesis driven overview of this topic, we conclude that although gene-environment interaction studies have highlighted the importance of serotonin transporter polymorphism in modulating glucocorticoid release, there is compelling evidence that age, gender and ethnicity are significant factors too contributing to the equation. We conclude too that the way early life events modulate an individual's stress reactivity as a function of serotonin transporter polymorphism is comparable between species. These data provide a rationale for the design of new, prospective translational studies into sex-specific gene-environment interactions across the lifespan with the goal of improving preventative efforts and optimizing (personalized) treatment in stress-related psychopathologies.

Radiological imaging of teratological fetuses: what can we learn?

OBJECTIVES: To determine the advantages of radiological imaging of a collection of full-term teratological fetuses in order to increase their scientific and educational value. BACKGROUND : Anatomical museums around the world exhibit full-term teratological fetuses. Unfortunately, these museums are regularly considered as "morbid cabinets". Detailed dysmorphological information concerning the exhibited specimens is often lacking. Moreover, fetuses with severe and complex congenital anomalies are frequently diagnosed incompletely, incorrectly or not at all. METHODS: In order to verify diagnoses and to enrich their educational and scientific value, we imaged 41 out of the 72 teratological specimens present in the collection of our Anatomy and Pathology Museum in Nijmegen (The Netherlands) by means of magnetic resonance imaging (MRI) and computed tomography (CT). Additionally, contemporary dysmorphological insights and 3D models are implemented in the teratology education of medical students and residents. CONCLUSIONS: Full-term teratological fetuses have become increasingly rare and deserve a prominent place in every anatomical museum; they are suitable for contemporary teratological research and education. Modern radiological techniques markedly enhance their scientific and didactic value. TEACHING POINTS: • To explore the scientific and educational potential of institutionalised teratological collections • To understand the additional value of radiological imaging in diagnosing teratological specimens • To learn about the specific settings of MRI parameters when scanning fixed specimens • To recognise specific internal dysmorphology in several congenital anomalies.

Epigenetic programming of the neuroendocrine stress response by adult life stress.

The hypothalamic-pituitary-adrenal (HPA) axis is critically involved in the neuroendocrine regulation of stress adaptation, and the restoration of homeostasis following stress exposure. Dysregulation of this axis is associated with stress-related pathologies like major depressive disorder, post-traumatic stress disorder, panic disorder and chronic anxiety. It has long been understood that stress during early life can have a significant lasting influence on the development of the neuroendocrine system and its neural regulators, partially by modifying epigenetic regulation of gene expression, with implications for health and well-being in later life. Evidence is accumulating that epigenetic plasticity also extends to adulthood, proposing it as a mechanism by which psychological trauma later in life can long-lastingly affect HPA axis function, brain plasticity, neuronal function and behavioural adaptation to neuropsychological stress. Further corroborating this claim is the phenomenon that these epigenetic changes correlate with the behavioural consequences of trauma exposure. Thereby, epigenetic modifications provide a putative molecular mechanism by which the behavioural phenotype and transcriptional/translational potential of genes involved in HPA axis regulation can change drastically in response to environmental challenges, and appear an important target for treatment of stress-related disorders. However, improved insight is required to increase their therapeutic (drug) potential. Here, we provide an overview of the growing body of literature describing the epigenetic modulation of the (primarily neuroendocrine) stress response as a consequence of adult life stress and interpret the implications for, and the challenges involved in applying this knowledge to, the identification and treatment of stress-related psychiatric disorders.

A comparative study of vascular injection fluids in fresh-frozen and embalmed human cadaver forearms.

Over the years, various vascular injection products have been developed to facilitate anatomical dissections. This study aimed to compare the most commonly used vascular injection products in fresh-frozen and formalin-embalmed cadaver specimens. An overview of the properties, advantages and limitations of each substance was given, and a comparison of vascular infusion procedures in both preservation methods was made. A literature search was performed in order to identify the most commonly used vascular injection products. Acrylic paint, latex, gelatin, silicone, Araldite F and Batson's No. 17 were selected for the study. One fresh-frozen and one embalmed cadaver forearm were infused with each injection product according to a uniform protocol. The curing time, skin- and subcutaneous tissue penetration, degree of filling of the arterial tree, extravasations, consistency of the injected vessels during dissection, and the costs of each injection fluid were noted. There was a large variation between the injection fluids in processing- and curing time, colour intensity, flexibility, fragility, elasticity, strength, toxicity and costs. All fluids were suitable for infusion. The penetration of injection fluid into the skin and subcutaneous tissue was significantly better in fresh-frozen specimens (P = 0.002 and P = 0.009, respectively), with significantly smaller branches casted (P = 0.004). Vascular infusion of fresh-frozen cadaver specimens results in a significantly better filled coloured arterial tree, enabling more detail to be achieved and smaller branches casted. The biomechanical properties of fresh-frozen soft tissues are less affected compared with formalin fixation. All the injection fluids studied are suitable for vascular infusion, but their different properties ensure that certain products and procedures are more suitable for specific study purposes.

Early life adversity and serotonin transporter gene variation interact at the level of the adrenal gland to affect the adult hypothalamo-pituitary-adrenal axis.

The short allelic variant of the serotonin transporter (5-HTT) promoter-linked polymorphic region (5-HTTLPR) has been associated with the etiology of major depression by interaction with early life stress (ELS). Furthermore, 5-HTTLPR has been associated with abnormal functioning of the stress-responsive hypothalamo-pituitary-adrenal (HPA) axis. Here, we examined if, and at what level, the HPA-axis is affected in an animal model for ELS × 5-HTTLPR interactions. Heterozygous and homozygous 5-HTT knockout rats and their wild-type littermates were exposed daily at postnatal days 2-14 to 3 h of maternal separation. When grown to adulthood, plasma levels of adrenocorticotropic hormone (ACTH), and the major rat glucocorticoid, corticosterone (CORT), were measured. Furthermore, the gene expression of key HPA-axis players at the level of the hypothalamus, pituitary and adrenal glands was assessed. No 5-HTT genotype × ELS interaction effects on gene expression were observed at the level of the hypothalamus or pituitary. However, we found significant 5-HTT genotype × ELS interaction effects for plasma CORT levels and adrenal mRNA levels of the ACTH receptor, such that 5-HTT deficiency was associated under control conditions with increased, but after ELS with decreased basal HPA-axis activity. With the use of an in vitro adrenal assay, naïve 5-HTT knockout rats were furthermore shown to display increased adrenal ACTH sensitivity. Therefore, we conclude that basal HPA-axis activity is affected by the interaction of 5-HTT genotype and ELS, and is programmed, within the axis itself, predominantly at the level of the adrenal gland. This study therefore emphasizes the importance of the adrenal gland for HPA-related psychiatric disorders.

Abnormal fat distribution in PMM2-CDG.

We hypothesize that abnormal fat distribution, a common feature of PMM2-CDG, is associated with abnormal perinatal hormone regulation. We assessed 32 cases with PMM2-CDG, for the comorbidity of hypoglycemia/hyperinsulinism and fat pads. Ninety percent of patients with hypoketotic hypoglycemia and/or hyperinsulinism had abnormal fat distribution, while normoglycemic patients showed this feature in 50% of the cases. This statistically significant difference suggests an etiological role of the insulin receptor in developing abnormal fat distribution in PMM2-CDG.

Orexinergic innervation of urocortin1 and cocaine and amphetamine regulated transcript neurons in the midbrain centrally projecting Edinger-Westphal nucleus.

Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger-Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either.

Nesfatin-1; implication in stress and stress-associated anxiety and depression.

Nesfatin-1, derived from an 82-amino-acid peptide precursor protein nucleobindin-2 (NUCB2), is a highly conserved peptide across mammalian species. Initial functional and neuroanatomical studies on NUCB2/nesfatin-1 in the central nervous system have supported a role for NUCB2/nesfatin-1 as a novel satiety molecule. In recent years, however, it has become apparent that this neuropeptide is involved in various other processes, one of which is the stress response. Stress-associated activation of NUCB2/nesfatin-1 neurons, together with nesfatin-1's central actions in the brain, is indicative of its significance in the stress adaptation response. Interestingly, increasing body of evidence implicates also NUCB2/nesfatin-1 in various forms of stress-associated psychopathologies, such as anxiety and depression. In this review, we will outline evidence that has significantly broadened our understanding of the biological significance of NUCB2/nesfatin-1 far beyond to be only a hypothalamic peptide with potent anorexigenic actions. NUCB2/nesfatin-1 neurons in the brain seem to emerge as novel, integral regulators of the stress adaptation response.

Cannabinoid modulation of midbrain urocortin 1 neurones during acute and chronic stress.

Neurones in the centrally projecting Edinger-Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour.

The amygdala, a relay station for switching on and off pain.

Neuropathic pain is strongly associated with mood disorders like anxiety and depression. Corticotropin-releasing factor (CRF) plays a prominent role in these disorders as it is up-regulated in limbic structures such as the amygdala, upon experimentally induced neuropathy. This review discusses recent literature on the role of CRF in pain processing and highlights the amygdala as a potential hot spot in supraspinal descending pain control. Many studies have demonstrated analgesic effects of CRF following local and systemic administration, but more recently also hyperalgesic effects were shown upon endogenous amygdalar CRF increase or by blocking the CRF type 1 receptor (CRFR1). On the basis of the reviewed literature, we postulate a central mechanism for pain control in which the amygdala plays a critical role by switching on and off chronic pain. In this mechanism, upon pain stimuli, CRFR1 in the amygdala is activated by CRF to induce hyperalgesia. When the activated CRFR1 is internalized (pain initiation), it triggers the translocation of the cytoplasmic CRF type 2 receptor (CRFR2) to the plasma membrane. Here, CRFR2 can be recruited by either high (pharmacological) concentrations of CRF or by endogenous CRFR2 ligands, the urocortins, leading to analgesia (pain termination). This on-off switching of pain is completed by redistribution of the CRF receptors to their initial activity state. We furthermore propose that in neuropathic pain, this mechanism is dysregulated and causes a state of permanent hyperalgesia, and present an integrative (patho)physiological model for the way disturbed CRF receptor signalling in the amygdala could initiate neuropathic pain.

The behavioral phenotype of pituitary adenylate-cyclase activating polypeptide-deficient mice in anxiety and depression tests is accompanied by blunted c-Fos expression in the bed nucleus of the stria terminalis, central projecting Edinger-Westphal nucleus, ventral lateral septum, and dorsal raphe nucleus.

Pituitary adenylate-cyclase activating polypeptide (PACAP) has been implicated in the (patho)physiology of stress-adaptation. PACAP deficient (PACAP(-/-)) mice show altered anxiety levels and depression-like behavior, but little is known about the underlying mechanisms in stress-related brain areas. Therefore, we aimed at investigating PACAP(-/-) mice in light-dark box, marble burying, open field, and forced swim paradigms. We also analyzed whether the forced swim test-induced c-Fos expression would be affected by PACAP deficiency in the following stress-related brain areas: magno- and parvocellular paraventricular nucleus of the hypothalamus (PVN); basolateral (BLA), medial (MeA), and central (CeA) amygdaloid nuclei; ventral (BSTv), dorsolateral (BSTdl), dorsomedial (BSTdm), and oval (BSTov) nuclei of the bed nucleus of stria terminalis; dorsal (dLS) and ventral parts (vLS) of lateral septal nucleus, central projecting Edinger-Westphal nucleus (EWcp), dorsal (dPAG) and lateral (lPAG) periaqueductal gray matter, dorsal raphe nucleus (DR). Our results revealed that PACAP(-/-) mice showed greatly reduced anxiety and increased locomotor activity compared with wildtypes. In forced swim test PACAP(-/-) mice showed increased depression-like behavior. Forced swim exposure increased c-Fos expression in all examined brain areas in wildtypes, whereas this was markedly blunted in the DR, EWcp, BSTov, BSTdl, BSTv, PVN, vLS, dPAG, and in the lPAG of PACAP(-/-) mice vs. wildtypes, strongly suggesting their involvement in the behavioral phenotype of PACAP(-/-) mice. PACAP deficiency did not influence the c-Fos response in the CeA, MeA, BSTdm, and dLS. Therefore, we propose that PACAP exerts a brain area-specific effect on stress-induced neuronal activation and it might contribute to stress-related mood disorders.

Experimental neuropathy increases limbic forebrain CRF.

Neuropathic pain is often accompanied by stress, anxiety and depression. Although there is evidence for involvement of corticotropin-releasing factor (CRF), the detailed neuronal basis of these pain-related mood alterations is unknown. This study shows that peripheral mononeuropathy was accompanied by changes in limbic forebrain CRF, but did not lead to changes in the functioning of the hypothalamo-pituitary-adrenal axis and the midbrain Edinger-Westphal centrally projecting (EWcp) neuron population, which play main roles in the organism's response to acute pain. Twenty-four days after chronic constriction injury (CCI) of the rat sciatic nerve, the oval bed nucleus of the stria terminalis (BSTov) contained substantially more Crf mRNA as did the central amygdala (CeA), which, in addition, possessed more CRF. In contrast, Crf mRNA and CRF contents of the hypothalamic paraventricular nucleus (PVN) were unaffected by CCI. Similarly, EWcp neurons, producing the CRF family member urocortin 1 (Ucn1) and constitutively activated by various stressors including acute pain, did not show an effect of CCI on Ucn1 mRNA or Ucn1. Also, the immediate early gene products cFos and deltaFosB in the EWcp were unaffected by CCI. These results indicate that neuropathic pain does not act via the HPA-axis or the EWcp, but includes a main role of Crf in the limbic system, which is in clear contrast to stressors like acute and chronic pain, which primarily act on the PVN and the EWcp.

Glycosylation defects underlying fetal alcohol spectrum disorder: a novel pathogenetic model. "When the wine goes in, strange things come out" - S.T. Coleridge, The Piccolomini.

Fetal alcohol spectrum disorder (FASD) is an umbrella term used to describe the craniofacial dysmorphic features, malformations, and disturbances in growth, neurodevelopment and behavior occurring in individuals prenatally exposed to alcohol. Fetal alcohol syndrome (FAS) represents the severe end of this spectrum. Many pathophysiological mechanisms have hitherto been proposed to account for the disrupted growth and morphogenesis seen in FAS. These include impaired cholesterol-modification of the Sonic hedgehog morphogen, retinoic acid deficiency, lipoperoxidative damage due to alcohol-induced reactive oxygen species combined with reduced antioxidant defences, and malfunctioning cell adhesion molecules. In this report, we propose a completely novel concept regarding the pathogenesis of FAS. Based on our observation that transferrin isoelectric focusing (TIEF) - the most widely used screening tool for congenital disorders of glycosylation (CDG) - was transiently abnormal in a newborn with FAS and a confirmed maternal history of gestational alcohol abuse, we came to believe that FAS exemplifies a congenital disorder of glycosylation secondary to alcohol-inflicted disruption of (N-linked) protein glycosylation. Various pieces of evidence were found in the literature to substantiate this hypothesis. This observation implies, among others, that one might need to consider the possibility of maternal alcohol consumption in newborns with transient glycosylation abnormalities. We also present an integrated pathophysiological model of FAS, which incorporates all existing theories mentioned above as well as our novel concept. This model highlights the pivotal role of disrupted isoprenoid metabolism in the origination of FAS.

Differential responses of corticotropin-releasing factor and urocortin 1 to acute pain stress in the rat brain.

It has been hypothesized that corticotropin-releasing factor (CRF) and its related neuropeptide urocortin 1 (Ucn1) play different roles in the initiation and adaptive phases of the stress response, which implies different temporal dynamics of these neuropeptides in response to stressors. We have tested the hypothesis that acute pain stress (APS) differentially changes the dynamics of CRF expression in the paraventricular nucleus of the hypothalamus (PVN), oval subdivision of the bed nucleus of the stria terminalis (BSTov) and central amygdala (CeA), and the dynamics of Ucn1 expression in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW). Thirty minutes after APS, induced by a formalin injection into the left hind paw, PVN, BSTov, CeA and npEW all showed a peak in cFos mRNA expression that was followed by a robust increase in cFos protein-immunoreactivity, indicating a rapid increase in (immediate early) gene expression in all four brain nuclei. CRF-dynamics, however, were affected by APS in a brain nucleus-specific way: in the PVN, CRF-immunoreactivity was minimal at 60 min after APS and concomitant with a marked increase in plasma corticosterone, whereas in the BSTov not CRF peptide but CRF mRNA peaked at 60 min, and in the CeA a surge of CRF peptide occurred as late as 240 min. The npEW differed from the other centers, as Ucn1 mRNA and Ucn1 peptide peaked at 120 min. These results support our hypothesis that each of the four brain centers responds to APS with CRF/Ucn1 dynamics that are specific as to nature and timing. In particular, we propose that CRF in the PVN plays a major role in the initiation phase, whereas Ucn1 in the npEW may act in the later, termination phase of the adaptation response to APS.

Stress-related changes in the activity of cocaine- and amphetamine-regulated transcript and nesfatin neurons in the midbrain non-preganglionic Edinger-Westphal nucleus in the rat.

Cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1/nucleobindin 2 (NUCB2) are assumed to play a role in feeding and adaptation to stress. Both peptides are highly expressed in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW), a center implicated in the regulation of stress adaptation and in the pathogenesis of stress-induced brain disorders, in a sex-specific manner. The present study was undertaken to test whether CART and nesfatin are involved in these actions of the npEW in the rat. Acute restraint and chronic variable mild stress were used. Following stress, physiological parameters (serum corticosterone levels, body, adrenal and thymus weights) were determined, CART and nesfatin-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (1) Acute stress resulted in an increase in serum corticosterone levels that was higher in females; (2) In males, data on corticosterone and body weight gain and in females, data on body weight gain revealed an effect of chronic stress; (3) Both acute and chronic stress activated npEW neurons expressing CART and nesfatin-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and nesfatin-LI in both males and females; (5) Neither acute nor chronic stress had an effect on CART and NUCB2 mRNA contents of npEW neurons in either sex. Taken together, our data suggest that CART and nesfatin are involved in the response of npEW neurons to chronic stress.

Depressive behaviour in children diagnosed with a mitochondrial disorder.

A higher incidence of depression has been described in adults with primary oxidative phosphorylation disease. We evaluated the psychological characteristics of eighteen non-retarded pediatric patients diagnosed with a disorder of the oxidative phosphorylation. We found significantly higher rate of withdrawn, depressive behaviour compared to population norm scores, to children with other types of inborn errors of metabolism and also in comparison to patients with Sotos syndrome. The occurrence of depressive behaviour showed no correlation with the degree of mitochondrial dysfunction. These findings support the hypothesis that mood disorders could be associated to abnormal cerebral energy metabolism.