Identification of nicotine-seeking and avoiding larval zebrafish using a new three-choice behavioral assay.

Introduction: Nicotine dependence is one of the main causes of preventable diseases in the United States. Nicotine-seeking and avoidance behavioral assays in larval zebrafish could be used for identifying potential new pharmacotherapeutics in an early phase of drug discovery and could facilitate the identification of genes and genomic variations associated with nicotine-seeking and avoidance behavior. Methods: A new three-choice behavioral assay has been developed for the identification of nicotine-seeking and avoiding larval zebrafish. The three choices are represented by three compartments of a gradient maze. Video-recording and subsequent quantitative analysis of the swimming track was carried out using EthovisionXT (Noldus). Results: Three behavioral phenotypes could be identified. Nicotine-seeking larval zebrafish occupied nicotine compartments for longer periods and entered the nicotine-containing compartments most frequently. Nicotine-avoiders spent most of the cumulative time in the water compartment or entered the water compartment most frequently. Non-seekers remained in the center compartment for most of the time. In the gradient maze, about 20-30% of larval zebrafish had a preference for low nicotine concentrations whereas nicotine avoidance was stronger at higher nicotine concentrations. Lower concentrations of nicotine (0.63 µM, 6.3 µM) resulted in higher percentages of nicotine seekers whereas high nicotine concentrations (63 µM, 630 µM) resulted in higher percentages of nicotine avoiders. Pre-treatment of larval zebrafish with nicotine slightly increased the percentage of nicotine avoiders at lower nicotine concentrations. Treatment with varenicline strongly increased the percentage of nicotine avoiders at lower nicotine concentrations. Conclusion: The results show that larval zebrafish have individual preferences for nicotine that could change with drug treatment. The three-choice gradient maze assay for larval zebrafish provides a new testing paradigm for studying the molecular and cellular mechanisms of nicotine action and the discovery of potential new pharmacotherapeutics for the treatment of smoking cessation.

Reactivity of the Oxytocinergic and Neuroendocrine System Following the Adult Attachment Projective Picture System in Men of Recent Fatherhood: Results from an Exploratory Pilot Study with a Cross-Sectional Design.

The attachment representation (AR) of individuals affects emotional and cognitive information processes and is considered an important modulating factor of neuroendocrine stress responses. The neuropeptide oxytocin is studied as one biomolecular component underpinning this modulation. A validated procedure used in attachment-related research is the Adult Attachment Projective Picture System (AAP). To date, only a limited number of studies investigated oxytocin and neuroendocrine reactivity in the context of an attachment-related stimulus similar to the APP. In this pilot study, N = 26 men of recent fatherhood were exposed to the AAP to classify AR and to investigate salivary changes in oxytocin, cortisol and dehydroepiandrosterone (DHEA) after AAP stimulation. We observed increased oxytocin levels in response to AAP exposure, and this increase was more pronounced in fathers with unresolved/disorganized AR. No significant changes in cortisol and DHEA concentrations were observed in response to AAP administration. Interestingly, differences in basal cortisol levels (before the AAP) also depended on AR classification. Here, the group of men with unresolved/disorganized AR showed higher levels of cortisol compared to fathers with organized AR. To conclude, the finding of increased salivary oxytocin levels in response to the AAP further indicates its validity as an instrument to stimulate the attachment system.

Associations between childhood maltreatment and DNA methylation of the oxytocin receptor gene in immune cells of mother-newborn dyads.

The neuropeptide oxytocin (OXT) and its receptor (OXTR) modulate interpersonal relationships, particularly mother-child interactions. DNA methylation (DNAm) changes of the OXTR gene were observed in individuals who experienced Childhood Maltreatment (CM). A modulatory role of single nucleotide polymorphisms (SNP) within OXTR in association with CM on the regulation of OXTR was also postulated. Whether these CM-induced epigenetic alterations are biologically inherited by the offspring remains unknown. We thus investigated possible intergenerational effects of maternal CM exposure on DNAm and OXTR gene expression, additionally accounting for the possible influence of three SNP: rs53576 and rs2254298 (OXTR gene), and rs2740210 (OXT gene). We used the Childhood Trauma Questionnaire to classify mothers into individuals with (CM+) or without CM (CM-). Maternal peripheral immune cells were isolated from venous blood (N = 117) and fetal immune cells from the umbilical cord (N = 113) after parturition. DNA methylation was assessed using MassARRAY. Taqman assays were performed for genotyping and gene expression analyses. Among mothers, CM was not associated with OXTR mean methylation or gene expression. However, four CpG sites showed different methylation levels in CM- compared to CM+. In mothers, the OXTR rs53576 and OXT rs2740210 allelic variations interacted with CM load on the OXTR mean methylation. Maternal and newborns' mean methylation of OXTR were positively associated within CM- dyads, but not in CM+ dyads. We show gene×environment interactions on the epigenetic regulation of the oxytocinergic signaling and show the intergenerational comparability of the OXTR DNAm might be altered in infants of CM+ mothers.

Systemic Catecholaminergic Deficiency in Depressed Patients with and without Coronary Artery Disease.

BACKGROUND: Stress and depression are known to contribute to coronary artery disease (CAD) with catecholamines (CA), altering the balance to a pro- and anti-inflammatory stetting and potentially playing a key role in the underlying pathophysiology. This study aimed to elucidate the impact of social stress on the CA system and inflammation markers in patients suffering from CAD and depression. METHODS: 93 subjects were exposed to the Trier Social Stress Test (TSST). Based on the results of the depression subscale of the Hospital Anxiety and Depression Scale (HADS, German Version) and the presence/absence of CAD, they were divided into four groups. A total of 21 patients suffered from CAD and depression (+D+CAD), 26 suffered from CAD alone (-D+CAD), and 23 suffered from depression only (+D-CAD); another 23 subjects served as healthy controls (-D-CAD). Subjects were registered at 09:00 AM at the laboratory. A peripheral venous catheter was inserted, and after a 60-min-resting period, the TSST was applied. Prior to and 5, 15, 30, and 60 min after the stress test, plasma epinephrine, norepinephrine, and dopamine concentrations (High Performance Liquid Chromatography (HPLC)) were measured together with the inflammation markers interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). High-sensitive C-reactive protein (hs-CRP, Enzyme-linked Immunosorbent Assay (ELISA)) was measured prior to TSST. RESULTS: (+D-CAD) and (+D+CAD) patients showed significantly lower epinephrine and dopamine levels compared to the (-D+CAD) and (-D-CAD) participants at baseline (prior to TSST). Over the whole measurement period after the TSST, no inter-group difference was detected. Partial correlation (controlling for age, gender and Body Mass Index (BMI)) revealed a significant direct relation between MCP-1 and norepinephrine (r = 0.47, p = 0.03) and MCP-1 and epinephrine (r = 0.46, p = 0.04) in patients with -D+CAD at rest. CONCLUSIONS: The stress response of the CA system was not affected by depression or CAD, whereas at baseline we detected a depression-related reduction of epinephrine and dopamine release independent of CAD comorbidity. Reduced norepinephrine and dopamine secretion in the central nervous system in depression, known as 'CA-deficit hypothesis', are targets of antidepressant drugs. Our results point towards a CA-deficit in the peripheral nervous system in line with CA-deficit of the central nervous system and CA exhaustion in depression. This might explain somatic symptoms such as constipation, stomach pain, diarrhoea, sweating, tremor, and the influence of depression on the outcome of somatic illness such as CAD.

Maternal Separation Induces Long-Term Alterations in the Cardiac Oxytocin Receptor and Cystathionine γ-Lyase Expression in Mice.

We recently showed that blunt chest trauma reduced the expression of the myocardial oxytocin receptor (Oxtr), which was further aggravated by genetic deletion of the H2S-producing enzyme cystathionine γ-lyase (CSE). Exogenous H2S supplementation restored myocardial Oxtr expression under these conditions. Early life stress (ELS) is a risk factor for cardiovascular disease by affecting vascular and heart structures. Therefore, we tested the hypotheses that (i) ELS affects cardiac Oxtr and CSE expressions and (ii) Oxtr and CSE expression patterns depend on the duration of stress exposure. Thus, two stress paradigms were compared: long- and short-term separation stress (LTSS and STSS, respectively). Cardiac Oxtr expression was differentially affected by the two stress paradigms with a significant reduction after LTSS and a significant increase after STSS. CSE expression, which was significantly reduced in Oxtr-/- knockout hearts, was downregulated and directly related to Oxtr expression in LTSS hearts (r = 0.657, p = 0.012). In contrast, CSE expression was not related to Oxtr upregulation in STSS. Plasma Oxt levels were not affected by either ELS paradigm. The coincidence of LTSS-induced reduction of cardiac Oxtr and reduced CSE expression may suggest a novel pathophysiological link between early life adversities and increased risk for the development of cardiovascular disorders in adulthood.

Maternal separation and contact to a stranger more than reunion affect the autonomic nervous system in the mother-child dyad: ANS measurements during Strange Situation Procedure in mother-child dyad.

BACKGROUND: The autonomic nervous system (ANS) processes underlying attachment-related mother-child interactions are not well understood. We aimed to describe and compare the responsivity of the ANS for mothers and their infants during the different phases of the Strange Situation Procedure (SSP). METHODS: Continuous measurements of the sympathetic (SNS) and parasympathetic (PNS) branches of the ANS were obtained simultaneously in 91 mothers and their infants (range 10-15 months). Heart rate (HR), respiratory sinus arrhythmia (RSA), pre-ejection period (PEP) and left ventricular ejection time (LVET), were calculated for the baseline period (e1) and seven subsequent episodes (e2-e8) of the SSP. RESULTS: The largest difference between the mother and infant was during e7, when the stranger went into the room where the infant was while the mother waited outside the room. Mothers showed reduced SNS-activity or stress reduction while the child showed PNS withdrawal or increased stress response. Additionally, LVET was found to be a marker for SNS changes in the one-year-old infant during SSP. CONCLUSION: Mothers and infants showed different stress-related ANS responses during e7. Since this study showed that simultaneous measurement of ANS responses in mother-child dyads during the SSP is feasible, future studies can assess both mother and child stress responses in different contexts. The measure of LVET may be a valid SNS-reactivity measure in the one-year-olds. Since the separation episode e7 led to the strongest ANS responses, future studies might assess stress responses in more normative circumstances, such as child care programs.

Reduced caregiving quality measured during the strange situation procedure increases child's autonomic nervous system stress response.

BACKGROUND: Dysfunctional maternal behavior has been shown to lead to disturbances in infant's regulatory capacities and alterations in vagal reactivity. We aim to investigate the autonomic nervous system (ANS) response of the child during the strange situation procedure (SSP) in relation to the quality of maternal behavior. METHODS: Twelve month after birth, 163 mother-child-dyads were investigated during the SSP. Heart rate (HR) and both, the parasympathetic branch (PNS) via the respiratory sinus arrhythmia (RSA) and the sympathetic branch (SNS) via the left ventricular ejection time (LVET) of the ANS were continuously determined during the SSP using electrocardiogram (ECG) and impedance cardiogram (ICG) measures. Maternal behavior was assessed by using the AMBIANCE measure. RESULTS: The ANS response in infants of mothers with disruptive behavior compared to infants of non-disruptive mothers was significantly altered during the SSP: HR increased especially when infants of disruptive mothers were alone with the stranger (F (1, 161) = 4.15, p = .04) with a significant vagal withdrawal when being in contact with the stranger despite of presence of the mother (F (1, 161) = 5.11, p = .03) and a significant increase in vagal tone during final reunion (F (1, 161) = 3.76, p = .05). HR increase was mainly based on a decrease in LVET (F (1, 161) = 4.08, p = .05) with a maximum infant's HR when the stranger came into the room instead of the mother. CONCLUSION: Both, SNS and PNS branches of the child are significantly altered in terms of an ANS imbalance, especially during contract to a stranger, in relation to dysfunctional maternal behavior. Our findings suggest the importance of supporting high quality caregiving that enables the infant to adapt adequately to stressful interpersonal situations which is likely to promote later health.

Are Circulating Tumor Cells (CTCs) Ready for Clinical Use in Breast Cancer? An Overview of Completed and Ongoing Trials Using CTCs for Clinical Treatment Decisions.

In recent years, breast cancer treatment has become increasingly individualized. Circulating tumor cells (CTCs) have the potential to move personalized medicine another step forward. The prognostic relevance of CTCs has already been proven both in early and metastatic breast cancer. In addition, there is evidence that changes in CTC numbers during the course of therapy can predict treatment response. Thus, CTCs are a suitable tool for repeated treatment monitoring through noninvasive liquid biopsy. The next step is to evaluate how this information can be used for clinical decision making with regard to the extension, modification, or abandonment of a treatment regimen. This review will summarize the completed and ongoing clinical trials using CTC number or phenotype for treatment decisions. Based on current knowledge, CTCs can be regarded as a useful prognostic and predictive marker that is well suited for both risk stratification and treatment monitoring in breast cancer patients. However, there is still the need to provide sufficient and unequivocal evidence for whether CTCs may indeed be used to guide treatment decisions in everyday clinical practice. The results of the ongoing trials described in this review are eagerly awaited to answer these important questions.

Hierarchical optimization for the efficient parametrization of ODE models.

Motivation: Mathematical models are nowadays important tools for analyzing dynamics of cellular processes. The unknown model parameters are usually estimated from experimental data. These data often only provide information about the relative changes between conditions, hence, the observables contain scaling parameters. The unknown scaling parameters and corresponding noise parameters have to be inferred along with the dynamic parameters. The nuisance parameters often increase the dimensionality of the estimation problem substantially and cause convergence problems. Results: In this manuscript, we propose a hierarchical optimization approach for estimating the parameters for ordinary differential equation (ODE) models from relative data. Our approach restructures the optimization problem into an inner and outer subproblem. These subproblems possess lower dimensions than the original optimization problem, and the inner problem can be solved analytically. We evaluated accuracy, robustness and computational efficiency of the hierarchical approach by studying three signaling pathways. The proposed approach achieved better convergence than the standard approach and required a lower computation time. As the hierarchical optimization approach is widely applicable, it provides a powerful alternative to established approaches. Availability and implementation: The code is included in the MATLAB toolbox PESTO which is available at http://github.com/ICB-DCM/PESTO. Supplementary information: Supplementary data are available at Bioinformatics online.

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells.

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system.

History of child maltreatment and telomere length in immune cell subsets: Associations with stress- and attachment-related hormones.

Experiencing maltreatment during childhood can have long-lasting consequences for both mental and physical health. Immune cell telomere length (TL) shortening might be one link between child maltreatment (CM) experiences and adverse health outcomes later in life. While the stress hormone cortisol has been associated with TL attrition, the attachment-related hormone oxytocin may promote resilience. In 15 mothers with and 15 age- and body mass index-matched mothers without CM, we assessed TL in peripheral blood mononuclear cells and selected immune cell subsets (monocytes, naive, and memory cytotoxic T cells) by quantitative fluorescence in situ hybridization, as well as peripheral cortisol and oxytocin levels. Memory cytotoxic T cells showed significantly shorter TL in association with CM, whereas TL in monocytes and naive cytotoxic T cells did not significantly differ between the two groups. Across both groups, cortisol was negatively associated with TL, while oxytocin was positively associated with TL in memory cytotoxic T cells. These results indicate that long-lived memory cytotoxic T cells are most affected by the increased biological stress state associated with CM. Keeping in mind the correlational and preliminary nature of the results, the data suggest that cortisol may have a damaging and oxytocin a protective function on TL.

PESTO: Parameter EStimation TOolbox.

Summary: PESTO is a widely applicable and highly customizable toolbox for parameter estimation in MathWorks MATLAB. It offers scalable algorithms for optimization, uncertainty and identifiability analysis, which work in a very generic manner, treating the objective function as a black box. Hence, PESTO can be used for any parameter estimation problem, for which the user can provide a deterministic objective function in MATLAB. Availability and implementation: PESTO is a MATLAB toolbox, freely available under the BSD license. The source code, along with extensive documentation and example code, can be downloaded from https://github.com/ICB-DCM/PESTO/. Contact: jan.hasenauer@helmholtz-muenchen.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Effects of the Adult Attachment Projective Picture System on Oxytocin and Cortisol Blood Levels in Mothers.

Oxytocin, a small neuropeptide of nine amino acids, has been characterized as the "hormone of affiliation" and is stimulated, for instance, in mothers when interacting with their offspring. Variations in maternal oxytocin levels were reported to predict differences in the quality of care provided by mothers. In this study, the Adult Attachment Projective Picture System (AAP) as a valid measure to assess attachment representations was used as an activating attachment-related stimulus. We investigated whether the AAP induces a release of oxytocin in mothers with a secure attachment representation and a stress-related cortisol response in mothers with an insecure attachment representation. Therefore, pre-post effects of AAP administration on plasma oxytocin and serum cortisol levels were investigated in n = 44 mothers 3 months after parturition. Oxytocin levels increased from pre to post in the significant majority of 73% participants (p = 0.004) and cortisol decreased in the significant majority of 73% participants (p = 0.004). Interestingly, no association between alterations in oxytocin and cortisol were found; this suggests taking a model of two independent processes into considerations. These results show that the AAP test procedure induces an oxytocin response. Concerning the results within the four AAP representation subgroups, our hypothesis of a particularly strong increase in oxytocin in secure mothers was not confirmed; however, in secure mothers we observed a particularly strong decrease in cortisol. Effect sizes are reported, allowing the replication of results in a larger study with sufficient sample size to draw final conclusions with respect to differences in OT and cortisol alterations depending on attachment representation. When interpreting the results, one should keep in mind that this study investigated lactating mothers. Thus, the generalizability of results is limited and future studies should investigate non-lactating healthy females as well as males and include a control stimulus condition.