Every day we are exposed to a large set of appetitive food cues, mostly of high caloric, high carbohydrate content. Environmental factors like food cue exposition can impact eating behavior, by triggering anticipatory endocrinal responses and reinforcing the reward value of food. Additionally, it has been shown that eating behavior is largely influence by neuroendocrine factors. Energy homeostasis is of great importance for survival in all animal species. It is challenged under the state of food deprivation which is considered to be a metabolic stressor. Interestingly, the systems regulating stress and food intake share neural circuits. Adrenal glucocorticoids, as cortisol, and the pancreatic hormone insulin have been shown to be crucial to maintain catabolic and anabolic balance. Cortisol and insulin can cross the blood-brain barrier and interact with receptors distributed throughout the brain, influencing appetite and eating behavior. At the same time, these hormones have an important impact on the stress response. The aim of the current work is to broaden the knowledge on reward related food cue processing. With that purpose, we studied how food cue processing is influenced by food deprivation in women (in different phases of the menstrual cycle) and men. Furthermore, we investigated the impact of the stress/metabolic hormones, insulin and cortisol, at neural sites important for energy metabolism and in the processing of visual food cues. The Chapter I of this thesis details the underlying mechanisms of the startle response and its application in the investigation of food cue processing. Moreover, it describes the effects of food deprivation and of the stress-metabolic hormones insulin and cortisol in reward related processing of food cues. It explains the rationale for the studies presented in Chapter II-IV and describes their main findings. A general discussion of the results and recommendations for future research is given. In the study described in Chapter II, startle methodology was used to study the impact of food deprivation in the processing of reward related food cues. Women in different phases of the menstrual cycle and men were studied, in order to address potential effects of sex and menstrual cycle. All participants were studied either satiated or food deprived. Food deprivation provoked enhanced acoustic startle (ASR) response during foreground presentation of visual food cues. Sex and menstrual cycle did not influence this effect. The startle pattern towards food cues during fasting can be explained by a frustrative nonreward effect (FNR), driven by the impossibility to consume the exposed food. In Chapter III, a study is described, which was carried out to explore the central effects of insulin and cortisol, using continuous arterial spin labeling to map cerebral blood flow patterns. Following standardized periods of fasting, male participants received either intranasal insulin, oral cortisol, both, or placebo. Intranasal insulin increased resting regional cerebral blood flow in the putamen and insular cortex, structures that are involved in the regulation of eating behavior. Neither cortisol nor interaction effects were found. These results demonstrate that insulin exerts an action in metabolic centers during resting state, which is not affected by glucocorticoids. The study described in Chapter IV uses a similar pharmacological manipulation as the one presented in Chapter III, while assessing processing of reward related food cues through the startle paradigm validated in Chapter II. A sample of men was studied during short-term food deprivation. Considering the importance of both cortisol and insulin in glucose metabolism, food pictures were divided by glycemic index. Cortisol administration enhanced ASR during foreground presentation of "high glycemic" food pictures. This result suggests that cortisol provokes an increase in reward value of high glycemic food cues, which is congruent with previous research on stress and food consumption. This thesis gives support to the FNR hypothesis towards food cues during states of deprivation. Furthermore, it highlights the potential effects of stress related hormones in metabolism-connected neuronal structures, and in the reward related mechanisms of food cue processing. In a society marked by increased food exposure and availability, alongside with increased stress, it is important to better understand the impact of food exposition and its interaction with relevant hormones. This thesis contributes to the knowledge in this field. More research in this direction is needed.

There is a lot of evidence for the impact of acute glucocorticoid treatment on hippocampus-dependent explicit learning and memory (memory for facts and events). But there have been few studies, investigating the effect of glucocorticoids on implicit learning and memory. We conducted three studies with different methodology to investigate the effect of glucocorticoids on different forms of implicit learning. In Study 1, we investigated the effect of cortisol depletion on short-term habituation in 49 healthy subjects. 25 participants received oral metyrapone (1500 mg) to suppress endogenous cortisol production, while 24 controls received oral placebo. Eye blink electromyogram (EMG) responses to 105 dB acoustic startle stimuli were assessed. Effective endogenous cortisol suppression had no effect on short-term habituation of the startle reflex, but startle eye blink responses were significantly increased in the metyrapone group. The latter findings are in line with previous human studies, which have shown that excess cortisol, sufficient to fully occupy central nervous system (CNS) corticosteroid receptors, may reduce startle eye blink. This effect may be mediated by CNS mechanisms controlling cortisol feedback. In Study 2, we investigated delay or trace eyeblink conditioning in a patient group with a relative hypocortisolism (30 patients with fibromyaligia syndrome/FMS) compared to 20 healthy control subjects. Conditioned eyeblink response probability was assessed by EMG. Morning cortisol levels, ratings of depression, anxiety and psychosomatic complaints as well as general symptomatology and psychological distress were assessed. As compared to healthy controls FMS patients showed lower morning cortisol levels, and trace eyeblink conditioning was facilitated whereas delay eyeblink conditioning was reduced. Cortisol measures correlate significantly only with trace eyeblink conditioning. Our results are in line with studies of pharmacologically induced hyper- and hypocortisolism, which affected trace eyeblink conditioning. We suggest that endocrine mechanisms affecting hippocampus-mediated forms of associative learning may play a role in the generation of symptoms in these patients.rnIn Study 3, we investigated the effect of excess cortisol on implicit sequence learning in healthy subjects. Oral cortisol (30 mg) was given to 29 participants, whereas 31 control subjects received placebo. All volunteers performed a 5-choice serial reaction time task (SRTT). The reaction speed of every button-press was determined and difference-scores were calculated as a proof of learning. Compared to the control group, we found a delayed learning in the cortisol group at the very beginning of the task. This study is the first human investigation, indicating impaired implicit memory function after exogenous administration of the stress hormone cortisol. Our findings support a previous neuroimaging study, which suggested that the medial temporal lobe (including the hippocampus) is also active in implicit sequence learning, but our results may also depend on the engagement of other brain structures.

In this thesis, three studies investigating the impact of stress on the protective startle eye blink reflex are reported. In the first study a decrease in prepulse inhibition of the startle reflex was observed after intravenous low dose cortisol application. In the second study a decrease in reflex magnitude of the startle reflex was observed after pharmacological suppression of endogenous cortisol production. In the third study, a higher reflex magnitude of the startle reflex was observed at reduced arterial and central venous blood pressure. These results can be interpreted in terms of an adaption to hostile environments.

The glucocorticoid (GC) cortisol, main mediator of the hypothalamic-pituitary-adrenal axis, has many implications in metabolism, stress response and the immune system. GC function is mediated mainly via the glucocorticoid receptor (GR) which binds as a transcription factor to glucocorticoid response elements (GREs). GCs are strong immunosuppressants and used to treat inflammatory and autoimmune diseases. Long-term usage can lead to several irreversible side effects which make improved understanding indispensable and warrant the adaptation of current drugs. Several large scale gene expression studies have been performed to gain insight into GC signalling. Nevertheless, studies at the proteomic level have not yet been made. The effects of cortisol on monocytes and macrophages were studied in the THP-1 cell line using 2D fluorescence difference gel electrophoresis (2D DIGE) combined with MALDI-TOF mass spectrometry. More than 50 cortisol-modulated proteins were identified which belonged to five functional groups: cytoskeleton, chaperones, immune response, metabolism, and transcription/translation. Multiple GREs were found in the promoters of their corresponding genes (+10 kb/-0.2 kb promoter regions including all alternative promoters available within the Database for Transcription Start Sites (DBTSS)). High quality GREs were observed mainly in cortisol modulated genes, corroborating the proteomics results. Differential regulation of selected immune response related proteins were confirmed by qPCR and immuno-blotting. All immune response related proteins (MX1, IFIT3, SYWC, STAT3, PMSE2, PRS7) which were induced by LPS were suppressed by cortisol and belong mainly to classical interferon target genes. Mx1 has been selected for detailed expression analysis since new isoforms have been identified by proteomics. FKBP51, known to be induced by cortisol, was identified as the strongest differentially expressed protein and contained the highest number of strict GREs. Genomic analysis of five alternative FKBP5 promoter regions suggested GC inducibility of all transcripts. 2D DIGE combined with 2D immunoblotting revealed the existence of several previously unknown FKBP51 isoforms, possibly resulting from these transcripts. Additionally multiple post-translational modifications were found, which could lead to different subcellular localization in monocytes and macrophages as seen by confocal microscopy. Similar results were obtained for the different cellular subsets of human peripheral blood mononuclear cells (PBMCs). FKBP51 was found to be constitutively phosphorylated with up to 8 phosphosites in CD19+ B lymphocytes. Differential Co-immunoprecipitation for cytoplasm and nucleus allowed us to identify new potential interaction partners. Nuclear FKBP51 was found to interact with myosin 9, whereas cytosolic FKBP51 with TRIM21 (synonym: Ro52, Sjögren`s syndrome antigen). The GR has been found to interact with THOC4 and YB1, two proteins implicated in mRNA processing and transcriptional regulation. We also applied proteomics to study rapid non-genomic effects of acute stress in a rat model. The nuclear proteome of the thymus was investigated after 15 min restraint stress and compared to the non-stressed control. Most of the identified proteins were transcriptional regulators found to be enriched in the nucleus probably to assist gene expression in an appropriate manner. The proteomic approach allowed us to further understand the cortisol mediated response in monocytes/macrophages. We identified several new target proteins, but we also found new protein variants and post-translational modifications which need further investigation. Detailed study of FKBP51 and GR indicated a complex regulation network which opened a new field of research. We identified new variants of the anti-viral response protein MX1, displaying differential expression and phosphorylation in the cellular compartments. Further, proteomics allowed us to follow the very early effects of acute stress, which happen prior to gene expression. The nuclear thymocyte proteome of restraint stressed rats revealed an active preparation for subsequent gene expression. Proteomics was successfully applied to study differential protein expression, to identify new protein variants and phosphorylation events as well as to follow translocation. New aspects for future research in the field of cortisol-mediated immune modulation have been added.

There is considerable evidence for an association between chronic dysregulation of the hypothalamus-pituitary adrenal (HPA) axis, atrophy of the hippocampus (HC) and cognitive and mood changes in clinical populations and in aging. The present thesis investigated this relationship in young healthy male subjects. Special emphasis was put on measures of HC volume and function derived from structural and functional magnetic resonance imaging (MRI). Higher cortisol levels after awakening were observed in subjects with higher levels of depressive symptomatology. Larger HC volume was associated with higher cortisol levels after awakening and in response to acute stress, whereas cognitive performance was impaired in subjects with larger HC volumes. Hippocampal activation during picture encoding was reduced after stress induction, and positive associations between activation and cognitive performance before stress were not present anymore afterwards. The present findings underscore the importance of structural and functional brain imaging for psychoneuroendocrinological research. The investigation of the association between cortisol levels and hippocampal integrity in young healthy subjects elicited unexpected results and adds to the understanding of HPA dysfunction and HC atrophy in clinical and aged populations.