Sex Differences In Stress Response And Depression: Study On Postmortem Human Brain Material And Rats Exposed To Stress

Part Ⅰ Increased GAD65/67 expression in the hypothalamic suprachiasmatic nucleus in depression:A postmortem studyBackground:Depression, also known as affective disorders, mainly includes major depressive disorder(MDD)and bipolar disorder (BD). A key characteristics of depression is hyperactivity of the stress regulation system, the hypothalamo-pituitary-adrenal (HPA)-axis. Another major neuropathological characteristic of depression is the disturbance of the central circadian pace maker, the hypothalamic suprachiasmatic nucleus (SCN), which leads to a disturbed sleep-wakefulness cycle and eating disorder. Arginine vasopressin is one of the main neuropeptide in the SCN. Both direct and indirect neuronal projections from the SCN to the paraventricular nucleus (PVN) have been observed in the hypothalamus of animals or human beings, which indicates a interaction between these two structures. Moreover are both nuclei innervated by other hypothalamic systems such as the orexin system, indicating a disfuctioned hypothalamic neuroncircuits. Although most SCN neurons produce gamma-aminobutyric acid (GABA), changes in SCN GABA in depression remain so far unknown. In addition, it remains unclear the interaction between the SCN and the hypothalamic orexin system and their relationship with the PVN activity in depression. We aimed, therefore, to reveal the GABAergic alterations in the SCN in mood disorder patients, in relation to the changes of SCN arginine-vasopressin (AVP) and hypothalamic orexin expression.Methods:13 mood disorder patients (6 MDD and 7 BD patients) and 13 controls were used in the present study, which were well-matched for possible confounding factors including age, sex, clock time of death, season of death, tissue fixation time, and tissue storage time. The amount of glutamic acid decarboxylase (GAD) 65/67-immunoreactivity (ir), which are key enzymes for GABA synthesis, and AVP-ir were detected in the SCN using immunocytochemistry (ICC). The amount of SCN GAD67-mRNA was determined by in situ hybridization (ISH). Image analysis was used to obtain the content of the ICC or ISH signals.Results:AVP-ir neurons and nerve fibres were found to be distributed in the SCN, the PVN, the supraoptic nucleus (SON) and the accessory nuclei between the PVN and the SON. GAD65/67-ir nerve fibres were found to be intensively distributed in the thalamus and the hypothalamus including the SCN, the PVN and the SON. The GAD67-mRNA-ISH signal showed similar distribution but only in cell bodies. There was a significant 54% increase of SCN GAD65/67-ir and a significant 165% increase of SCN GAD67-mRNA in the mood disorder group (P= 0.044, and P= 0.029, respectively). In addition, there was a significant 155% increase of AVP-ir in female (P= 0.008) but not in male mood disorder patients. SCN-AVP-ir showed a significant negative correlation with age in the control group (Rho=-0.745, P= 0.003), which was also found in male-(Rho=-0.766, P= 0.027) but not in female mood disorder patients. Moreover, hypothalamic orexin-ir showed a positive correlation with SCN GAD-ir in male controls (Rho= 0.790, P= 0.020), while no such a correlation was found in female controls. No difference was found between MDD and BD patients.Conclusion:The strongly increased SCN GABA (shown as elevated GAD65/67 and GAD67-mRNA level) may be central to the disturbance of SCN function in depression. The abnormal expression of AVP-ir in SCN and hypothalamic orexin-ir in female mood disorder patients may both reflect the circadian disturbance and HPA hyperactivity and may contribute to the sex differences in the prevalence of depression.Part Ⅱ Sex differences in the stress response in Sprague-Dawley rats:changes in hypothalamus and peripheryBackground:Previous studies have shown that sex differences play an important role in depression, the basis of which is an excessive stress response. A hyperactive hypothalamo-pituitary-adrenal (HPA)-axis, a key system regulating the stress response, was observed in depression and excessive stress can lead to the occurrence and exacerbation of depression. We and other groups have also observed that sex hormones such as estradiol (E2) and testosterone (T) regulate HPA activity through their respective receptors, i.e. estrogen receptor a or P(ERa, ERβ and androgen receptor (AR), acting on the CRH gene promoter. Estrogens stimulate CRH gene transcription, thus activating the activity of HPA-axis, while androgens inhibit this process. These mechanisms may be essential for sex differences in the responses to stress and morbidity of depression in human. In order to further investigate the possible mechanisms involved in human depression in stressed animal models, we aimed to reveal if similar sex differences exist in the acute-or chronically-stressed rats, especially in hypothalamus. We aimed to study changes in plasma corticosterone (CORT) and sex hormones (T and E2) levels, as well as the hypothalamic mRNA-expression of stress-related molecules, including ERa, ERp\AR, aromatase (ARO)-the key enzyme that transforms androgen into estrogen, the CORT receptors, i.e. the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), and the neuropeptides CRH, arginine vasopressin (AVP), and oxytocin (OXT).Methods:Female Sprague-Dawley (SD) rats were randomly divided into groups: chronic unpredictable mild stress (CUMS), acute foot shock (FS) and controls, animals in all 3 groups were further sacrificed in either the proestrus or diestrus thus leading to 6 groups. Male SD rats were randomly divided into 3 groups:CUMS, FS and controls. Comparisons were also made for behavioral changes, i.e. open field test, sucrose preference test after the CUMS procedure was finished. Rats were then sacrificed and the hypothalamus and plasma samples were collected. Plasma corticosterone (CORT) levels were determined by enzyme-linked immunosorbent assay (ELISA), while testosterone (T) and estradiol (E2) levels determined by radioimmunoassay. Hypothalamic mRNA-expression of stress-related molecules were measured by quantitative reverse transcription PCR (Q-PCR).Results:CUMS resulted in disordered estrus cycles, i.e. in longer diestrus phases. Female CUMS rats showed more anxious- or depressive-like behaviors and more changes in hypothalamic stress-related molecules, including a significant increase in CRH-mRNA (p=0.05), decrease in MR-mRNA and GR-mRNA (P=0.003 and P= 0.001, respectively) compared with female control rats, while male CUMS rates showed significantly increased AVP-mRNA compared to male control rats (P< 0.001). In addition, stressed female rats showed larger increasing amplitudes of plasma CORT levels in relation to their baseline levels (18.7 times for CUMS and 34.3 times for FS) than stressed male rats (1.8 times for CUMS and 3.6 times for FS). Female rats also showed decreased E2 and T levels after FS and CUMS (P<0.039), while male FS rats showed increased E2 (P= 0.056) and male CUMS rats showed decreased T levels (P= 0.0047).Conclusion:Acute and chronic stress affect 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.Part III Effect of pentobarbital or isoflurane on acute stress response in ratBackground:The relationship between disordered stress responses and the etiology of depression has boosted studies on the stress responses in animal models. Stress-regulating systems such as the hypothalamo-pituitary-adrenal (HPA)-axis are hyperactive in depression. We and other groups have observed that sex steroids such as testosterone (T) and estradiol (E2), and neuropeptides such as arginine vasopressin (AVP) and oxytocin (OXT), play a crucial role in the regulation of the stress response. Notably, anesthesia administration before sacrificing animals is a common practice in stress-related studies, but the effect of anesthesia on the results remains understudied. Anesthetics affect brain activity by inhibition of excitatory receptors, including N-methyl-D-aspartate (NMDA) receptor subunit NR2B and the neuronal-type nicotinic acetylcholine receptor (nnAChR), and by stimulation of inhibitory receptors such as GABAA receptor (GABAAR). So far it is unclear whether the often used anesthetics such as intraperitoneal (i.p.) injection of sodium-pentobarbital or isoflurane inhalation may affect the results obtained in the study of an acute stress reaction. We aimed to reveal the interference of different anesthetics, i.e. intraperitoneal (i.p.) sodium-pentobarbital injection or isoflurane inhalation, with the acute stress responses in rats, including plasma corticosterone (CORT), T and E2 levels, the hypothalamic mRNA levels of CRH, AVP and OXT, and the frontal lobe mRNA levels of NMDA-NR2B, GABAAR and nnAChR.Methods:Rats were randomly divided into foot shock (FS) and non-stressed control groups, and further grouped according to the sacrificing procedure:direct decapitation, decapitation after i.p. sodium-pentobarbital injection, or isoflurane inhalation (6 groups in total). There was also a non-stressed group sacrificed by decapitation following i.p. saline injection. Samples of plasma, the hypothalamus and the frontal lobe were collected. Plasma CORT levels were determined by enzyme-linked immunosorbent assay, plasma T by radioimmunoassay, plasma E2 by enzyme immunoassay, mRNA expression of the hypothalamic stress-related molecules corticotropin-releasing hormone (CRH), AVP and OXT, and frontal lobe stress-related molecule NR2B, GABAAR and nnAChR-mRNA were measured using quantitative reverse transcription PCR(Q-PCR).Results:FS significantly increased plasma CORT levels (P< 0.001) in direct decapitation and isoflurane groups, while this stress response’disappeared’(P= 0.132) following i.p. sodium-pentobarbital injection. In control animals, both the injection of saline and pentobarbital caused a significant increase of plasma CORT (P= 0.008, P= 0.004). Neither the sex hormone levels nor the mRNA expression of stress-related molecules in the brain showed significant differences among the groups (P> 0.132).Conclusion:The injection of the anesthetic compound rather than the compound itself may cause extra stress which increases the plasma CORT levels. This makes the difference between stress and control group’disappear, thus interfering the CORT results. However there was no interference with plasma sex hormone levels nor with the brain mRNA expression. Isoflurane inhalation leaves the stress response intact and is also optimal from an ethical point of view for future mechanistic studies on the stress response.