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INTRODUCTION: Women are more vulnerable to Alzheimer's disease (AD) than men. The entorhinal cortex (EC) is one of the earliest structures affected in AD. We identified in cognitively intact elderly different molecular changes in the EC in relation to age. METHODS: Changes in 12 characteristic molecules in relation to age were determined by quantitative immunohistochemistry or in situ hybridization in the EC. They were arbitrarily grouped into sex steroid-related molecules, markers of neuronal activity, neurotransmitter-related molecules, and cholinergic activity-related molecules. RESULTS: The changes in molecules indicated increasing local estrogenic and neuronal activity accompanied by a higher and faster hyperphosphorylated tau accumulation in women's EC in relation to age, versus a mainly stable local estrogenic/androgenic and neuronal activity in men's EC. DISCUSSION: EC employs a different neurobiological strategy in women and men to maintain cognitive function, which seems to be accompanied by an earlier start of AD in women. HIGHLIGHTS: Local estrogen system is activated with age only in women's entorhinal cortex (EC). EC neuronal activity increased with age only in elderly women with intact cognition. Men and women have different molecular strategies to retain cognition with aging. P-tau accumulation in the EC was higher and faster in cognitively intact elderly women.

AIMS Women are more vulnerable to Alzheimer's disease (AD) than men. We investigated (i) whether and at what age the AD hallmarks, that is, β-amyloid (Aβ) and hyperphosphorylated Tau (p-Tau) show sex differences; and (ii) whether such sex differences may occur in cognitively intact elderly individuals. METHODS We first analysed the entire post-mortem brain collection of all non-demented 'controls' and AD donors from our Brain Bank (245 men and 403 women), for the presence of sex differences in AD hallmarks. Second, we quantitatively studied possible sex differences in Aβ, Aβ42 and p-Tau in the entorhinal cortex of well-matched female (n = 31) and male (n = 21) clinically cognitively intact elderly individuals. RESULTS Women had significantly higher Braak stages for tangles and amyloid scores than men, after 80 years. In the cognitively intact elderly, women showed higher levels of p-Tau, but not Aβ or Aβ42, in the entorhinal cortex than men, and a significant interaction of sex with age was found only for p-Tau but not Aβ or Aβ42. CONCLUSIONS Enhanced p-Tau in the entorhinal cortex may play a major role in the vulnerability to AD in women.

Hypocretin (also called orexin) regulates various functions, such as sleep-wake rhythms, attention, cognition, and energy balance, which show significant changes in schizophrenia (SCZ). We aimed to identify alterations in the hypocretin system in SCZ patients. We measured plasma hypocretin-1 levels in SCZ patients and healthy controls and found significantly decreased plasma hypocretin-1 levels in SCZ patients, which was mainly due to a significant decrease in female SCZ patients compared with female controls. In addition, we measured postmortem hypothalamic hypocretin-1-immunoreactivity (ir), ventricular cerebrospinal fluid (CSF) hypocretin-1 levels, and hypocretin receptor (Hcrt-R) mRNA expression in the superior frontal gyrus (SFG) in SCZ patients and controls We observed a significant decrease in the amount of hypothalamic hypocretin-1 ir in SCZ patients, which was due to decreased amounts in female but not male patients. Moreover, Hcrt-R2 mRNA in the SFG was decreased in female SCZ patients compared with female controls, while male SCZ patients showed a trend of increased Hcrt-R1 mRNA and Hcrt-R2 mRNA expression compared with male controls. We conclude that central hypocretin neurotransmission is decreased in SCZ patients, especially female patients, and this is reflected in the plasma.

Gender identity (an individual's perception of being male or female) and sexual orientation (heterosexuality, homosexuality, or bisexuality) are programmed into our brain during early development. During the intrauterine period in the second half of pregnancy, a testosterone surge masculinizes the fetal male brain. If such a testosterone surge does not occur, this will result in a feminine brain. As sexual differentiation of the brain takes place at a much later stage in development than sexual differentiation of the genitals, these two processes can be influenced independently of each other and can result in gender dysphoria. Nature produces a great variability for all aspects of sexual differentiation of the brain. Mechanisms involved in sexual differentiation of the brain include hormones, genetics, epigenetics, endocrine disruptors, immune response, and self-organization. Furthermore, structural and functional differences in the hypothalamus relating to gender dysphoria and sexual orientation are described in this review. All the genetic, postmortem, and in vivo scanning observations support the neurobiological theory about the origin of gender dysphoria, i.e., it is the sizes of brain structures, the neuron numbers, the molecular composition, functions, and connectivity of brain structures that determine our gender identity or sexual orientation. There is no evidence that one's postnatal social environment plays a crucial role in the development of gender identity or sexual orientation.

The quality of postmortem hypothalamus research depends strongly on a thorough clinical investigation and documentation of the patient's disorder and therapies. In addition, a systematic and professional neuropathological investigation of the entire brain of both the cases and the controls is absolutely crucial. In the experience of the Netherlands Brain Bank (NBB), about 20% of the clinical neurological diagnoses, despite being made in first rate clinics, have to be revised or require extra diagnoses after a complete and thorough neuropathologic review by the NBB. The neuropathology examination may reveal for instance that the elderly "controls" already have preclinical neurodegenerative alterations. In postmortem studies, the patient and control groups must be matched for as many as possible of the known confounding factors. This is necessary to make the groups as similar as possible, except for the topic being investigated. Confounding factors are present (i) before, (ii) during, and (iii) after death. They are, respectively: (i) genetic background, systemic diseases, duration and gravity of illness, medicines and addictive compounds used, age, sex, gender identity, sexual orientation, clock- and seasonal time of death, and lateralization; (ii) agonal state, stress of dying; and (iii) postmortem delay, freezing procedures, fixation, and storage time. Agonal state is generally estimated by measuring the pH of the brain. However, there are disorders in which pH is lower as a part of the disease process. Because of the large number of potentially confounding factors that differ according to, for instance, brain area and disease, a brain bank should have a large number of controls at its disposal for appropriate matching. If matching fails for some confounders, the influence of the confounders may be determined by statistical methods, such as analysis of variance or the regression models.

There are no specific structural neuropathological hallmarks found in the brain of mood disorders. Instead, there are molecular, functional and structural alterations reported in many brain areas. The neurodevelopmental underpinning indicated the presence of various genetic and developmental risk factors. The effect of genetic polymorphisms and developmental sequalae, some of which may start in the womb, result in functional changes in a network mediated by neurotransmitters and neuropeptides, which make the emotion- and stress-related brain systems more vulnerable to stressful events. This network of stress-related neurocircuits consists of, for instance, brainstem nuclei, the amygdala, habenula, prefrontal cortex and hypothalamus. Various nuclei of the hypothalamus form indeed one of the crucial hubs in this network. This structure concerns not only the hypothalamo-pituitary-adrenal (HPA) axis that integrate the neuro-endocrine-immune responses to stress, but also other hypothalamic nuclei and systems that play a key role in the symptoms of depression, such as disordered day-night rhythm, lack of reward feelings, disturbed eating, sex, and disturbed cognitive functions. The present review will focus on the changes in the human hypothalamus in depression, with the HPA axis in the center. We will discuss the inordinate network of neurotransmitters and neuropeptides involved, with the hope to find the most vulnerable neurobiological systems and the possible development of tailor-made treatments for mood disorders in the future. Keywords: The hypothalamo-pituitary-adrenal axis, Glutamate, GABA, Neuropeptide, Sex differences

Alzheimer’s disease (AD) is characterized by decreased neuronal activity and atrophy, while hyperactivity of neurons seems to make them resistant to aging and neurodegeneration, a phenomenon which we have paraphrased as ‘use it or lose it’. Our hypothesis proposes that (1) during their functioning, neurons are damaged; (2) accumulation of damage that is not repaired is the basis of aging; (3) the vulnerability to AD is determined by the genetic background and the balance between the amount of damage and the efficiency of repair, and (4) by stimulating the brain, repair mechanisms are stimulated and cognitive reserve is increased, resulting in a decreased rate of aging and risk for AD. Environmental stimulating factors such as bilingualism/multilingualism, education, occupation, musical experience, physical exercise, and leisure activities have been reported to reduce the risk of dementia and decrease the rate of cognitive decline, although methodological problems are present.

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

Understanding the genetic architecture of gene expression and splicing in human brain is critical to unlocking the mechanisms of complex neuropsychiatric disorders like schizophrenia (SCZ). Large-scale brain transcriptomic studies are based primarily on populations of European (EUR) ancestry. The uniformity of mono-racial resources may limit important insights into the disease etiology. Here, we characterized brain transcriptional regulatory architecture of East Asians (EAS; n=151), identifying 3,278 expression quantitative trait loci (eQTL) and 4,726 spliceQTL (sQTL). Comparing these to PsychENCODE/BrainGVEX confirmed our hypothesis that the transcriptional regulatory architecture in EAS and EUR brains align. Furthermore, distinctive allelic frequency and linkage disequilibrium impede QTL translation and gene-expression prediction accuracy. Integration of eQTL/sQTL with genome-wide association studies reveals common and novel SCZ risk genes. Pathway-based analyses showing shared SCZ biology point to synaptic and GTPase dysfunction as a prospective pathogenesis. This study elucidates the transcriptional landscape of the EAS brain and emphasizes an essential convergence between EAS and EUR populations.

Negative symptoms in schizophrenia strongly contribute to poor functional outcomes, however its pathogenesis is still unclear. Here, we found that histamine H1 receptor (H1R) expression in basal forebrain (BF) cholinergic neurons was decreased in patients with schizophrenia having negative symptoms. Deletion of H1R gene in cholinergic neurons in mice resulted in functional deficiency of cholinergic projections from the BF to the prefrontal cortex and in the formation of sensorimotor gating deficit, social impairment and anhedonia-like behavior. These behavioral deficits can be rescued by re-expressing H1R or by chemogenetic activation of cholinergic neurons in the BF. Direct chemogenetic inhibition of BF cholinergic neurons produced such behavioral deficits and also increased the susceptibility to hyperlocomotion. Our results suggest that the H1R deficiency in BF cholinergic neurons is critical for sensorimotor gating deficit, social impairments and anhedonia-like behavior. This finding may help to understand the genetic and biochemical bases of negative symptoms in schizophrenia.

The quality of postmortem hypothalamus research depends strongly on a thorough clinical investigation and documentation of the patient's disorder and therapies. In addition, a systematic and professional neuropathological investigation of the entire brain of both the cases and the controls is absolutely crucial. In the experience of the Netherlands Brain Bank (NBB), about 20% of the clinical neurological diagnoses, despite being made in first rate clinics, have to be revised or require extra diagnoses after a complete and thorough neuropathologic review by the NBB. The neuropathology examination may reveal for instance that the elderly "controls" already have preclinical neurodegenerative alterations. In postmortem studies, the patient and control groups must be matched for as many as possible of the known confounding factors. This is necessary to make the groups as similar as possible, except for the topic being investigated. Confounding factors are present (i) before, (ii) during, and (iii) after death. They are, respectively: (i) genetic background, systemic diseases, duration and gravity of illness, medicines and addictive compounds used, age, sex, gender identity, sexual orientation, clock- and seasonal time of death, and lateralization; (ii) agonal state, stress of dying; and (iii) postmortem delay, freezing procedures, fixation, and storage time. Agonal state is generally estimated by measuring the pH of the brain. However, there are disorders in which pH is lower as a part of the disease process. Because of the large number of potentially confounding factors that differ according to, for instance, brain area and disease, a brain bank should have a large number of controls at its disposal for appropriate matching. If matching fails for some confounders, the influence of the confounders may be determined by statistical methods, such as analysis of variance or the regression models.

Gender identity (an individual's perception of being male or female) and sexual orientation (heterosexuality, homosexuality, or bisexuality) are programmed into our brain during early development. During the intrauterine period in the second half of pregnancy, a testosterone surge masculinizes the fetal male brain. If such a testosterone surge does not occur, this will result in a feminine brain. As sexual differentiation of the brain takes place at a much later stage in development than sexual differentiation of the genitals, these two processes can be influenced independently of each other and can result in gender dysphoria. Nature produces a great variability for all aspects of sexual differentiation of the brain. Mechanisms involved in sexual differentiation of the brain include hormones, genetics, epigenetics, endocrine disruptors, immune response, and self-organization. Furthermore, structural and functional differences in the hypothalamus relating to gender dysphoria and sexual orientation are described in this review. All the genetic, postmortem, and in vivo scanning observations support the neurobiological theory about the origin of gender dysphoria, i.e., it is the sizes of brain structures, the neuron numbers, the molecular composition, functions, and connectivity of brain structures that determine our gender identity or sexual orientation. There is no evidence that one's postnatal social environment plays a crucial role in the development of gender identity or sexual orientation.

Stress-related disorders, such as mood disorders and posttraumatic stress disorder (PTSD), are more common in women than in men. This sex difference is at least partly due to the organizing effect of sex steroids during intrauterine development, while activating or inhibiting effects of circulating sex hormones in the postnatal period and adulthood also play a role. Such effects result in structural and functional changes in neuronal networks, neurotransmitters, and neuropeptides, which make the arousal- and stress-related brain systems more vulnerable to environmental stressful events in women. Certain brainstem nuclei, the amygdala, habenula, prefrontal cortex, and hypothalamus are important hubs in the stress-related neuronal network. Various hypothalamic nuclei play a central role in this sexually dimorphic network. This concerns not only the hypothalamus-pituitary-adrenal axis (HPA-axis), which integrates the neuro-endocrine-immune responses to stress, but also other hypothalamic nuclei and systems that play a key role in the symptoms of mood disorders, such as disordered day-night rhythm, lack of reward feelings, disturbed eating and sex, and disturbed cognitive functions. The present chapter focuses on the structural and functional sex differences that are present in the stress-related brain systems in mood disorders and PTSD, placing the HPA-axis in the center. The individual differences in the vulnerability of the discussed systems, caused by genetic and epigenetic developmental factors warrant further research to develop tailor-made therapeutic strategies.

Background There are currently no objective diagnostic biomarkers for major depressive disorder (MDD) due to the biological complexity of the disorder. The existence of blood-based biomarkers with high specificity would be convenient for the clinical diagnosis of MDD. Methods A comprehensive plasma proteomic analysis was conducted in a highly homogeneous cohort [7 drug-naive MDD patients and 7 healthy controls (HCs)], with bioinformatics analysis combined with machine learning used to screen candidate proteins. Verification of reproducibility and specificity was conducted in independent cohorts [60 HCs and 74 MDD, 42 schizophrenia (SZ) and 39 bipolar I disorder (BD-I) drug-naive patients]. Furthermore, verification of consistency was accomplished by proteomic analysis of postmortem brain tissue from 16 MDD patients and 16 HCs. Results: Levels of C-reactive protein (CRP), antithrombin III (ATIII), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and vitamin D-binding protein (VDB) were significantly higher in MDD patients, both in the discovery cohort and independent replication cohort. In comparison with SZ or BD-I patients, two proteins (VDB and ITIH4) were significantly elevated only in MDD patients. In addition, increased VDB and ITIH4 were observed consistently in both plasma and postmortem dorsolateral prefrontal cortex tissues of MDD patients. Furthermore, a panel consisting of all four plasma proteins was able to distinguish MDD patients from HCs or SZ or BD-I patients with the highest accuracy. Conclusion Plasma ITIH4 and VDB may be potential plasma biomarkers of MDD with high specificity. The four-protein panel is more suitable as a potential clinical diagnostic marker for MDD.

Stress-related disorders, such as mood disorders and posttraumatic stress disorder (PTSD), are more common in women than in men. This sex difference is at least partly due to the organizing effect of sex steroids during intrauterine development, while activating or inhibiting effects of circulating sex hormones in the postnatal period and adulthood also play a role. Such effects result in structural and functional changes in neuronal networks, neurotransmitters, and neuropeptides, which make the arousal- and stress-related brain systems more vulnerable to environmental stressful events in women. Certain brainstem nuclei, the amygdala, habenula, prefrontal cortex, and hypothalamus are important hubs in the stress-related neuronal network. Various hypothalamic nuclei play a central role in this sexually dimorphic network. This concerns not only the hypothalamus-pituitary-adrenal axis (HPA-axis), which integrates the neuro-endocrine-immune responses to stress, but also other hypothalamic nuclei and systems that play a key role in the symptoms of mood disorders, such as disordered day-night rhythm, lack of reward feelings, disturbed eating and sex, and disturbed cognitive functions. The present chapter focuses on the structural and functional sex differences that are present in the stress-related brain systems in mood disorders and PTSD, placing the HPA-axis in the center. The individual differences in the vulnerability of the discussed systems, caused by genetic and epigenetic developmental factors warrant further research to develop tailor-made therapeutic strategies.

Oxytocin (OXT), synthesized in the hypothalamic paraventricular nucleus (PVN) and then released into different brain areas, may play a crucial role in various behaviors and neuropsychiatric disorders, including depression. Testosterone has been proposed by clinical studies to have the opposite effect of oxytocin in these disorders. We began by studying, in the postmortem hypothalamus of fifteen patients with mood disorders and fifteen matched controls, the expression of OXT in the PVN by means of immunocytochemistry (ICC) and the co-localization of OXT and androgen receptor (AR) by means of double labeling ICC. Subsequently, the regulatory effect of AR on OXT gene expression was studied in vitro. We found a higher expression of PVN OXT in the mood disorder patients than in the control subjects, and observed a clear co-localization of AR in OXT-expressing neurons, both in the cytoplasm and in the nucleus. In addition, a significant decrease in OXT-mRNA levels was observed after pre-incubation of the SK-N-SH cells with testosterone. A further potential androgen-responsive element in the human OXT gene promotor was revealed by electrophoretic mobility shift assays and co-transfections in neuroblastoma cells. Finally, in vitro studies demonstrated that AR mediated the down-regulation of OXT gene expression. These results suggest that the fact that OXT and testosterone appear to have opposite effects in neuropsychiatric disorders might be based upon a direct inhibition of AR on OXT transcription, which may provide a novel target for therapeutic strategies in depression.Neuropsychopharmacology accepted article preview online, 27 April 2017. doi:10.1038/npp.2017.76.

Background Neurophysiological and behavioral processes regulated by hypocretin (orexin) are severely affected in depression. However, alterations in hypocretin have so far not been studied in the human brain. We explored the hypocretin system changes in the hypothalamus and cortex in depression from male and female subjects. Methods We quantified the differences between depression patients and well-matched controls, in terms of hypothalamic hypocretin-1 immunoreactivity (ir) and hypocretin receptors (Hcrtr-receptors)-mRNA in the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex. In addition, we determined the alterations in the hypocretin system in a frequently used model for depression, the chronic unpredictable mild stress (CUMS) rat. Results i) Compared to control subjects, the amount of hypocretin-immunoreactivity (ir) was significantly increased in female but not in male depression patients; ii) hypothalamic hypocretin-ir showed a clear diurnal fluctuation, which was absent in depression; iii) male depressive patients who had committed suicide showed significantly increased ACC Hcrt-receptor-2-mRNA expression compared to male controls; and iv) female but not male CUMS rats showed a highly significant positive correlation between the mRNA levels of corticotropin-releasing hormone and prepro-hypocretin in the hypothalamus, and a significantly increased Hcrt-receptor-1-mRNA expression in the frontal cortex compared to female control rats. Conclusions The clear sex-related change found in the hypothalamic hypocretin-1-ir in depression should be taken into account in the development of hypocretin-targeted therapeutic strategies., Highlights • Hypocretin (orexin) changes were studied in human postmortem brain in depression. • A clear sex-related change was found in the hypothalamic hypocretin-1-immunoreactivity in depression. • A rat depression model did not reflect the changes in the hypocretin system in the human brain in depression. The stress systems of depressed patients are put into a higher gear by genetic and developmental factors. Over-reaction of these systems to stressful environmental situations makes people vulnerable to depression and suicide. This is the first postmortem study on changes in a relatively novel stress system in depression, consisting of the hypothalamic hypocretin neurons and hypocretin receptors in the prefrontal cortex. A clear sex-related change was found in the hypothalamic hypocretin-1-immunoreactivity in depression. Evaluation of the hypocretin system in a frequently used depression animal model, i.e. chronic unpredictable mild stress rats, did not replicate changes found in the hypocretin systems in the human brain in depression.

In depression, disrupted circadian rhythms reflect abnormalities in the central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Although many SCN neurons are said to be GABAergic, it was not yet known whether and how SCN GABA changes occur in the SCN in depression. We, therefore, studied GABA in the SCN in relation to the changes in arginine vasopressin (AVP), which is one of the major SCN output systems. Postmortem hypothalamus specimens of 13 subjects suffering from depression and of 13 well-matched controls were collected. Quantitative immunocytochemistry was used to analyze the protein levels of glutamic acid decarboxylase (GAD)65/67 and AVP, and quantitative in situ hybridization was used to measure transcript levels of GAD67 in the SCN. There were a significant 58% increase of SCN GAD65/67-ir and a significant 169% increase of SCN GAD67-mRNA in the depression group. In addition, there were a significant 253% increase of AVP-ir in female depression subjects but not in male depression patients. This sex difference was supported by a re-analysis of SCN AVP-ir data of a previous study of our group. Moreover, SCN-AVP-ir showed a significant negative correlation with age in the control group and in the male, but not in the female depression group. Given the crucial role of GABA in mediating SCN function, our finding of increased SCN GABA expression may significantly contribute to the disordered circadian rhythms in depression. The increased SCN AVP-ir in female-but not in male-depression patients-may reflect the higher vulnerability for depression in women.

Our previous studies showed that the transcription factor early growth response-1 (EGR1) may play a role in keeping the brain cholinergic function intact in the preclinical stages of Alzheimer's disease (AD). In order to elucidate the mechanisms involved, we first performed data mining on our previous microarray study on postmortem human prefrontal cortex (PFC) for the changes in the expression of EGR1 and acetylcholinesterase (AChE) and the relationship between them during the course of AD. The study contained 49 patients, ranging from non-demented controls (Braak stage 0) to late AD patients (Braak stage VI). We found EGR1-mRNA was high in early AD and decreased in late AD stages, while AChE-mRNA was stable in preclinical AD and slightly decreased in late AD stages. A significant positive correlation was found between the mRNA levels of these two molecules. In addition, we studied the relationship between EGR1 and AChE mRNA levels in the frontal cortex of 3-12-months old triple-transgenic AD (3xTg-AD) mice. EGR1- and AChE-mRNA were lower in 3xTg-AD mice compared with wild type (WT) mice. A significant positive correlation between these two molecules was present in the entire group and in each age group of either WT or 3xTg-AD mice. Subsequently, AChE expression was determined following up- or down-regulating EGR1 in cell lines and the EGR1 levels were found to regulate AChE at both the mRNA and protein levels. Dual-luciferase assay and electrophoretic mobility shift assay in the EGR1-overexpressing cells were performed to determine the functionally effective binding sites of the EGR1 on the AChE gene promoter. We conclude that the EGR1 can upregulate AChE expression by a direct effect on its gene promoter, which may contribute significantly to the changes in cholinergic function in the course of AD. The 3xTg-AD mouse model only reflects later stage AD. This article is protected by copyright. All rights reserved.

The cholinergic nucleus basalis of Meynert, which is important for memory functions, shows neuronal activation ('up-phase') during the early stages of Alzheimer's disease and neurodegeneration ('down-phase') in later stages of Alzheimer's disease. MicroRNA-132 (miR-132) and the transcription factor early growth response-1 (EGR1) were proposed as possible candidate molecules regulating such an up-down activity pattern of the nucleus basalis of Meynert during the course of Alzheimer's disease, as they both show this up-down pattern of expression in the prefrontal cortex during the course of Alzheimer's disease. Not only do these two molecules stimulate synaptic activity and plasticity, they are also involved in Alzheimer's disease pathology and might, in addition, affect cholinergic function. In the nucleus basalis of Meynert, we investigated the expression of miR-132 and EGR1 along the entire course of Alzheimer's disease. Forty-nine post-mortem nucleus basalis of Meynert samples were studied, ranging from non-demented controls (Braak stage = 0) to late Alzheimer's disease patients (Braak stage = VI), and from clinical Reisberg scale 1 to 7. Each Braak stage contained seven samples, that were all well matched for confounding factors, i.e. age (range 58-91), sex, post-mortem delay, cerebrospinal fluid pH (as a measure for agonal state), APOE genotype, clock time of death, tissue fixation time, and tissue storage time. The alterations of these two molecules were studied over the course of Alzheimer's disease in relation to the expression of 4G8-stained amyloid-β, hyperphosphorylated tau stained by antibody AT8, neuronal fibrillary tangles and neuropil threads stained by silver, and in relation to alterations in choline acetyltransferase. We found that the expression of miR-132 and EGR1 in the nucleus basalis of Meynert was quite stable during the early stages of Alzheimer's disease and decreased significantly only during late Alzheimer's disease stages. In addition, miR-132 and EGR1 showed a significant positive correlation with choline acetyltransferase expression (r = 0.49, P < 0.001 and r = 0.61, P < 0.001), while choline acetyltransferase expression showed a significantly negative correlation with hyperphosphorylated tau (r = -0.33, P = 0.021) but no correlation with 4G8-stained amyloid-β. From the functional changes of miR-132 and EGR1 along the course of Alzheimer's disease we conclude: (i) that these two molecules may play a role in keeping the cholinergic function intact in early Alzheimer's disease stages; and (ii) that these molecules may contribute to the late neurodegeneration of this cholinergic nucleus.

Ion-exchange high performance liquid chromatography (HPLC) generally fails as a method to determine low levels of free amino acids (AAs) in body fluids. Here we present a modified reversed-phase HPLC (RP-HPLC) protocol for the determination of AAs in body fluids and its application in mood disorder patients. We improved a previous research protocol by modifying i) sample preparation, including deproteination, ii) derivitization, including derivating agent and condition, and iii) sample separation, which is mainly determined by the pH value, the components and the additives of the mobile phases. The combination of these modifications, together with fluorescence detection (FLD), allows sensitive and practical determination of free AA levels in body fluids of depressive patients. This protocol was validated by determining the postmortem cerebrospinal fluid (CSF) glutamic acid (Glu) and γ-aminobutyric acid (GABA) levels of 8 major depressive disorder (MDD) patients, 9 bipolar disorder (BD) patients, and 19 well-matched controls, while also testing the plasma and CSF AA levels of living MDD patients. CSF Glu and GABA levels were both significantly decreased in MDD but not in BD patients. The data indicate that this RP-HPLC-FLD protocol is applicable for detection of low levels of neuroactive AAs in body fluids, as well as for routine clinical applications.

Background Classic nuclear-initiated estrogen signaling stimulates corticotropin-releasing hormone (CRH) gene expression as a transcription factor. However, the possible mechanism by which membrane-initiated estrogen signaling (MIES) influences CRH expression remains unclear. There are indications that MIES may upregulate nitric oxide (NO) production through the phosphatidylinositol 3-hydroxy kinase (PI3K) and potentially through the mitogen-activated protein kinase (MAPK) pathway. Objectives We investigated the effect of MIES-mediated kinase pathways on CRH expression with or without NO synthesis. Method In SK-N-SH cell culture, estradiol-bovine serum albumin (E2-BSA) was used as the specific membrane estrogen receptor activator, with a specific NO donor, and/or inhibitors for NO synthase (NOS), PI3K, MAPK, protein kinase A (PKA), and protein kinase C (PKC). Results E2-BSA significantly increased NO and CRH levels in the medium and NOS1-mRNA levels in the cells. In addition, NO donor up-regulated CRH expression, while NOS-inhibitor down-regulated it. When the inhibitor of MAPK and/or the inhibitor of PI3K was added to the medium, only the latter appeared to significantly block the stimulating effect of E2-BSA on NO synthesis, and this was accompanied by an increased CRH expression in the medium. We further studied the effect of the MIES-PKC-mediated pathway on CRH expression, with or without NOS-inhibitor, while the MIES-PKA(-PI3K) pathway served as a control. We found that MIES-PKC upregulated CRH expression independent of NO synthesis. Conclusion MIES can efficiently upregulate CRH expression via various intracellular kinase pathways and may thus be a crucial component in the stress response.

The locus coeruleus (LC) has been studied in major depressive disorder (MDD) and bipolar disorder (BD). A major problem of immunocytochemical studies in the human LC is interference with the staining of the immunocytochemical end-product by the omnipresent natural brown pigment neuromelanin. Here, we used a multispectral method to untangle the two colors: blue immunocytochemical staining and brown neuromelanin. We found significantly increased tyrosine hydroxylase (TH) in the LC of MDD patients-thus validating the method-but not in BD patients, and we did not find significant changes in the receptor tyrosine-protein kinase ErbB4 in the LC in MDD or BD patients. We observed clear co-localization of ErbB4, TH, and neuromelanin in the LC neurons. The different stress-related molecular changes in the LC may contribute to the different clinical symptoms in MDD and BD.

Dysfunction of the noradrenergic (NE) neurons is implicated in the pathogenesis of bipolar disorder (BPD). ErbB4 is highly expressed in NE neurons, and its genetic variation has been linked to BPD; however, how ErbB4 regulates NE neuronal function and contributes to BPD pathogenesis is unclear. Here we find that conditional deletion of ErbB4 in locus coeruleus (LC) NE neurons increases neuronal spontaneous firing through NMDA receptor hyperfunction, and elevates catecholamines in the cerebrospinal fluid (CSF). Furthermore, Erbb4-deficient mice present mania-like behaviors, including hyperactivity, reduced anxiety and depression, and increased sucrose preference. These behaviors are completely rescued by the anti-manic drug lithium or antagonists of catecholaminergic receptors. Our study demonstrates the critical role of ErbB4 signaling in regulating LC-NE neuronal function, reinforcing the view that dysfunction of the NE system may contribute to the pathogenesis of mania-associated disorder., eLife digest Bipolar disorder is a mental illness that affects roughly 1 in 100 people worldwide. It features periods of depression interspersed with episodes of mania – a state of delusion, heightened excitation and increased activity. Evidence suggests that changes in a brain region called the locus coeruleus contribute to bipolar disorder. Cells within this area produce a chemical called norepinephrine, whose levels increase during mania and decrease during depression. But it is unclear exactly how norepinephrine-producing cells, also known as noradrenergic cells, contribute to bipolar disorder. The answer may lie in a protein called ErbB4, which is found within the outer membrane of many noradrenergic neurons. ErbB4 is active in both the developing and adult brain, and certain people with bipolar disorder have mutations in the gene that codes for the protein. Might changes in ErbB4 disrupt the activity of noradrenergic neurons? And could these changes increase the risk of bipolar disorder? To find out, Cao, Zhang et al. deleted the gene for ErbB4 from noradrenergic neurons in the locus coeruleus of mice. The mutant mice showed mania-like behaviors: compared to normal animals, they were hyperactive, less anxious, and consumed more of a sugary solution. Treating the mice with lithium, a medication used in bipolar disorder, reversed these changes and made the rodents behave more like non-mutant animals. Further experiments revealed that noradrenergic neurons in the mutant mice showed increased spontaneous activity. These animals also had more of the chemicals noradrenaline and dopamine in the fluid circulating around their brains and spinal cords. The results thus suggest that losing ErbB4 enhances the spontaneous firing of noradrenergic neurons in the locus coeruleus. This increases release of noradrenaline and dopamine, which in turn leads to mania-like behaviors. Future research should examine whether drugs that target ErbB4 could treat mania and improve the lives of people with bipolar disorder and related conditions.

The quality of postmortem research depends strongly on a thorough clinical investigation and documentation of the patient's disorder and therapies. In addition, a systematic and professional neuropathologic investigation of both cases and controls is absolutely crucial. In the experience of the Netherlands Brain Bank (NBB), about 20% of clinical neurologic diagnoses, despite being made in first-rate clinics, have to be revised or require an extra diagnosis after a complete and thorough review by the NBB. The neuropathology examination may reveal for instance that the "controls" already have preclinical neurodegenerative alterations. In postmortem studies the patient and control groups must be matched for as many of the known confounding factors as possible. This is necessary to make the groups as similar as possible, except for the topic being investigated. Confounding factors are present before, during, and after death. They are respectively: (1) genetic background, systemic diseases, duration and gravity of illness, medicines and addictive compounds used, age, sex, gender identity, sexual orientation, circadian and seasonal fluctuations, lateralization; (2) agonal state, stress of dying; and (3) postmortem delay, freezing procedures, fixation and storage time. Consequently, a brain bank should have a large number of controls at its disposal for appropriate matching. If matching fails for some confounders, then their influence may be determined by statistical methods such as analysis of variance or regression models.

Background Alterations in peripheral sex hormones may play an important role in sex differences in terms of stress responses and mood disorders. It is not yet known whether and how stress-related brain systems and brain sex steroid levels fluctuate in relation to changes in peripheral sex hormone levels, or whether the different sexes show different patterns. We aimed to investigate systematically, in male and female rats, the effect of decreased circulating sex hormone levels following gonadectomy on acute and chronic stress responses, manifested as changes in plasma and hypothalamic sex steroids and hypothalamic stress-related molecules. Method Experiment (Exp)-1: Rats (14 males, 14 females) were gonadectomized or sham-operated (intact); Exp-2: gonadectomized and intact rats (28 males, 28 females) were exposed to acute foot shock or no stressor; and Exp-3: gonadectomized and intact rats (32 males, 32 females) were exposed to chronic unpredictable mild stress (CUMS) or no stressor. For all rats, plasma and hypothalamic testosterone (T), estradiol (E2), and the expression of stress-related molecules were determined, including corticotropin-releasing hormone, vasopressin, oxytocin, aromatase, and the receptors for estrogens, androgens, glucocorticoids, and mineralocorticoids. Results Surprisingly, no significant correlation was observed in terms of plasma sex hormones, brain sex steroids, and hypothalamic stress-related molecule mRNAs (p > 0.113) in intact or gonadectomized, male or female, rats. Male and female rats, either intact or gonadectomized and exposed to acute or chronic stress, showed different patterns of stress-related molecule changes. Conclusion Diminished peripheral sex hormone levels lead to different peripheral and central patterns of change in the stress response systems in male and female rats. This has implications for the choice of models for the study of the different types of mood disorders which also show sex differences.

Sex differences play an important role in depression, the basis of which is an excessive stress response. We aimed at revealing the neurobiological sex differences in the same study in acute- and chronically-stressed rats. Female Sprague-Dawley (SD) rats were randomly divided into 6 groups: chronic unpredictable mild stress (CUMS), acute foot shock (FS) and controls, animals in all 3 groups were sacrificed in proestrus or diestrus. Male SD rats were randomly divided into 3 groups: CUMS, FS and controls. Comparisons were made of behavioral changes in CUMS and control rats, plasma levels of corticosterone (CORT), testosterone (T) and estradiol (E2), and of the hypothalamic mRNA-expression of stress-related molecules, i.e. estrogen receptor α and β, androgen receptor, aromatase, mineralocorticoid receptor, glucocorticoid receptor, corticotropin-releasing hormone, arginine vasopressin and oxytocin. CUMS resulted in disordered estrus cycles, more behavioral and hypothalamic stress-related molecules changes and a stronger CORT response in female rats compared with male rats. Female rats also showed decreased E2 and T levels after FS and CUMS, while male FS rats showed increased E2 and male CUMS rats showed decreased T levels. Stress affects the behavioral, endocrine and the molecular response of the stress systems in the hypothalamus of SD rats in a clear sexual dimorphic way, which has parallels in human data on stress and depression.

Compared to other monoamine neurotransmitters, information on the association between the histaminergic system and neuropsychiatric disorders is scarce, resulting in a lack of histamine-related treatment for these disorders. The current chapter tries to combine information obtained from genetic studies, neuroimaging, post-mortem human brain studies and cerebrospinal fluid measurements with data from recent clinical trials on histamine receptor agonists and antagonists, with a view to determining the possible role of the histaminergic system in neuropsychiatric disorders and to pave the way for novel histamine-related therapeutic strategies.

Sexual differentiation of the brain, gender identity (an individual's perception of being male or female), and sexual orientation (heterosexuality, homosexuality, bisexuality, and pedophilia) is orchestrated and determined in our brain during early development. Sexual differentiation of the brain is determined by a series of events, starting with the influence from the SRY gene on the Y chromosome, followed by the influence of sex hormones regulated by different mechanisms, through different paths and with different endpoints, including epigenetic modifications. These complex mechanisms ensure the variability, necessary from an evolutionary perspective; however, it also provides multiple points where it may be disrupted or modified, and therefore influence sexual behaviors. This chapter will review the literature regarding the genetic, postmortem, and in vivo scanning observations that support the neurobiological theory about the origin of our gender identity and sexual orientation. In humans, there is no evidence that one's postnatal social environment plays a crucial role in the development of gender identity and sexual orientation.

Compared to other monoamine neurotransmitters, information on the association between the histaminergic system and neuropsychiatric disorders is scarce, resulting in a lack of histamine-related treatment for these disorders. The current chapter tries to combine information obtained from genetic studies, neuroimaging, post-mortem human brain studies and cerebrospinal fluid measurements with data from recent clinical trials on histamine receptor agonists and antagonists, with a view to determining the possible role of the histaminergic system in neuropsychiatric disorders and to pave the way for novel histamine-related therapeutic strategies.