Christina Dalla

Chronic mild stress impact: Are females more vulnerable?

Despite the knowledge that women are more susceptible than men to stress-related mental illness, such as major depression, there is no comprehensive estimation of the role of gender in the detrimental effects of chronic stress that might cause depression. Sex differences regarding the association of behavioral parameters with serotonergic and hypothalamic-pituitary-adrenal axis activities were investigated in the chronic mild stress model of depression. Additionally, the impact of chronic mild stress exposure on an additional/novel short-term stressful procedure, such as the forced swim test was examined in male and female rats. Female rats were found to be more vulnerable to chronic mild stress and that was depicted with disruption of sucrose intake, decreases in open field activity, increased corticosterone levels, alteration in estrous cycle and decreased serotonergic activity in hippocampus and hypothalamus. On the contrary, in males the current chronic mild stress protocol elicited only behavioral changes, such as disruption in sucrose intake and decreased open field activity. Interestingly, in response to forced swim test, females previously subjected to chronic mild stress, were found to cope better by exhibiting increased active behavior in the second forced swim test session and higher hypothalamic serotonergic activity in comparison with corresponding males. On the other hand, males were more affected by previous chronic mild stress exposure and that was manifested by decreased active behavior in the first forced swim test session and increased corticosterone levels following second forced swim test session. These data indicate that although females are more vulnerable in the application of chronic mild stress than males, in response to an additional-novel stressor (forced swim test) they show better response. Therefore, both sex/gender and combination of stressful procedures should be carefully considered in the study of the pathophysiology of stress-related mental illnesses.

Sex differences in learning processes of classical and operant conditioning

Males and females learn and remember differently at different times in their lives. These differences occur in most species, from invertebrates to humans. We review here sex differences as they occur in laboratory rodent species. We focus on classical and operant conditioning paradigms, including classical eyeblink conditioning, fear-conditioning, active avoidance and conditioned taste aversion. Sex differences have been reported during acquisition, retention and extinction in most of these paradigms. In general, females perform better than males in the classical eyeblink conditioning, in fear-potentiated startle and in most operant conditioning tasks, such as the active avoidance test. However, in the classical fear-conditioning paradigm, in certain lever-pressing paradigms and in the conditioned taste aversion, males outperform females or are more resistant to extinction. Most sex differences in conditioning are dependent on organizational effects of gonadal hormones during early development of the brain, in addition to modulation by activational effects during puberty and adulthood. Critically, sex differences in performance account for some of the reported effects on learning and these are discussed throughout the review. Because so many mental disorders are more prevalent in one sex than the other, it is important to consider sex differences in learning when applying animal models of learning for these disorders. Finally, we discuss how sex differences in learning continue to alter the brain throughout the lifespan. Thus, sex differences in learning are not only mediated by sex differences in the brain, but also contribute to them.

Sex differences in the effects of two stress paradigms on dopaminergic neurotransmission.

Sex differences in behavioral and neurobiological responses to stress are considered to modulate the prevalence of some psychiatric disorders, including major depression. In the present study, we compared dopaminergic neurotransmission and behavior in response to two different stress paradigms, the Forced Swim Test (FST) and the Chronic Mild Stress (CMS). Male and female rats were subjected to one session of swim stress for two consecutive days (FST) or to a variety of mild stressors alternating for six weeks (CMS). Subsequently, the tissue levels of dopamine (DA) and its metabolites (HVA and DOPAC) in the hippocampus, the hypothalamus, the prefrontal cortex and the striatum were measured using high-performance liquid chromatography (HPLC). The ratios HVA/DA and DOPAC/DA were also calculated as indices of the dopaminergic activity. Results from the FST determined that males exhibited lower immobility, higher climbing duration and increased dopaminergic activity in the prefrontal cortex and the hippocampus compared to females. CMS induced alterations in sucrose intake in both sexes, while it only decreased dopaminergic activity in the prefrontal cortex of females. These findings show that FST and CMS have different effects on the dopaminergic activity of discrete brain regions depending on the sex of the animal. These data support the growing evidence that females display a differential response and adaptation to stress than males.

Females do not Express Learned Helplessness like Males do

Women are more likely than men to suffer from stress-related mental disorders, such as depression. In the present experiments, we identified sex differences in one of the most common animal models of depression, that of learned helplessness. Male and female rats were trained to escape a mild footshock each day for 7 days (controllable stress). Each rat was yoked to another rat that could not escape (uncontrollable stress), but was exposed to the same amount of shock. One day later, all stressed rats and unstressed controls were tested on a more difficult escape task in a different context. Most males exposed to uncontrollable stress did not learn to escape and were therefore helpless. In contrast, most females did learn to escape on the more difficult escape task, irrespective of whether they had been exposed to controllable or uncontrollable stress. The sex differences in helplessness behavior were not dependent on the presence of sex hormones in adulthood, because neither ovariectomy of females nor castration of males abolished them. The absence of helplessness in females was neither dependent on organizational effects of testosterone during the day of birth, because masculinized females did not express helplessness as adults. Thus, sex differences in helplessness behavior are independent of gonadal hormones in adulthood and testosterone exposure during perinatal development. Learned helplessness may not constitute a valid model for depressive behavior in women, at least as reflected by the response of female rats to operant conditioning procedures after stressful experience.

Estradiol rapidly activates male sexual behavior and affects brain monoamine levels in the quail brain.

Steroids are generally viewed as transcription factors binding to intracellular receptors and activating gene transcription. Rapid cellular effects mediated via non-genomic mechanisms have however been identified and one report showed that injections of estradiol rapidly stimulate chemoinvestigation and mounting behavior in castrated male rats. It is not known whether such effects take place in other species and what are the cellular underlying mechanisms. We show here that a single injection of estradiol (500 microg/kg) rapidly and transiently activates copulatory behavior in castrated male quail pre-treated with a dose of testosterone behaviorally ineffective by itself. The maximal behavioral effect was observed after 15 min. In a second experiment, the brain of all subjects was immediately collected after behavioral tests performed 15 min after injection. The preoptic area--hypothalamus (HPOA), hindbrain, telencephalon and cerebellum were isolated and monoamines measured by HPLC-ED. Estradiol increased levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA/serotonin ratios in the telencephalon and hindbrain independently of whether animals had mated or not. Estradiol also affected these measures in HPOA and cerebellum but this effect was correlated with the level of sexual activity so that significant effects of the treatment only appeared when sexual activity was used as a covariate. Interactions between estradiol effects and sexual activity were also observed for dopamine in the HPOA and for serotonin in the hindbrain and cerebellum. Together, these data demonstrate that a single estradiol injection rapidly activates male sexual behavior in quail and that this behavioral effect is correlated with changes in monoaminergic activity.

Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology.

We recently found that female aromatase knockout (ArKO) mice that are deficient in oestradiol due to a targeted mutation in the aromatase gene show deficits in sexual behaviour that cannot be corrected by adult treatment with oestrogens. We determined here whether these impairments are associated with changes in general levels of activity, anxiety or ‘depressive‐like’ symptomatology due to chronic oestrogen deficiency. We also compared the neurochemical profile of ArKO and wild‐type (WT) females, as oestrogens have been shown to modulate dopaminergic, serotonergic and noradrenergic brain activities. ArKO females did not differ from WT in spontaneous motor activity, exploration or anxiety. These findings are in line with the absence of major neurochemical alterations in hypothalamus, prefrontal cortex or striatum, which are involved in the expression of these behaviours. By contrast, ArKO females displayed decreased active behaviours, such as struggling and swimming, and increased passive behaviours, such as floating, in repeated sessions of the forced swim test, indicating that these females exhibit ‘depressive‐like’ symptoms. Adult treatment with oestradiol did not reverse the behavioural deficits observed in the forced swim test, suggesting that they may be due to the absence of oestradiol during development. Accordingly, an increased serotonergic activity was observed in the hippocampus of ArKO females compared with WT, which was also not reversed by adult oestradiol treatment. The possible organizational role of oestradiol on the hippocampal serotonergic system and the ‘depressive‐like’ profile of ArKO females provide new insights into the pathophysiology of depression and the increased vulnerability of women to depression.

Female rats learn trace memories better than male rats and consequently retain a greater proportion of new neurons in their hippocampi

Learning increases the survival of new cells that are generated in the hippocampal formation before the training experience, especially if the animal learns to associate stimuli across time [Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ (1999) Nat Neurosci 2:260–265]. All relevant studies have been conducted on male rats, despite evidence for sex differences in this type of learning. In the present study, we asked whether sex differences in learning influence the survival of neurons generated in the adult hippocampus. Male and female adult rats were injected with one dose of bromodeoxyuridine (BrdU; 200 mg/kg), to label one population of dividing cells. One week later, half of the animals were trained with a temporal learning task of trace eyeblink conditioning, while the other half were not trained. Animals were killed 1 day after training (12 days after the BrdU injection). Hippocampal tissue was stained for BrdU and a marker of immature neurons, doublecortin. Both sexes learned to emit the conditioned eyeblink response during the trace interval. As a consequence, more new neurons remained in their hippocampi than in sex-matched controls. In individual animals, the number of surviving cells correlated positively with asymptotic performance; those that expressed more learned responses retained more new neurons. However, animals that learned very well retained even more new cells if they required many trials to do so. Because females emitted more learned responses than males did, they retained nearly twice as many new cells per unit volume of tissue. This effect was most evident in the ventral region of the hippocampal formation. Thus, sex differences in learning alter the anatomical structure of the hippocampus. As a result, male and female brains continue to differentiate in adulthood.

Sex Differences in Response to Stress and Expression of Depressive-Like Behaviours in the Rat

Women are more susceptible than men to certain stress-related psychiatric disorders, such as depression. Preclinical studies aim to understand these sex differences by studying male and female rats in stress models. In this chapter, we review sex differences in behavioural aspects, as well as neurochemical and neurobiological findings derived from acute, repeated and chronic stress models. In particular, we focus on sex differences in depressive-like symptomatology expressed in the forced swim test, the chronic mild stress (CMS) and the learned helplessness models, the Flinders Sensitive Line rats (FSL), which is a genetic model of depression and in the lipopolysaccharide (LPS)-induced sickness behaviour, a putative inflammatory model of depression. Also, sex differences in stress effects on learning and memory parameters are discussed, because cognitive alterations are often seen in sex-differentiated psychiatric disorders. The observed behavioural alterations are often linked with abnormalities in the endophenotype, such as in hormonal, neurochemical, immune and neuroplasticity indices. From these data, it is clear that all stress models have strengths and limitations that need to be recognized in order to use them effectively in the investigation of sex differences in affective disorders.

Sex-related differential response to clomipramine treatment in a rat model of depression

Research in affective disorders is often performed without considering sex differences, although women are predominantly affected. Consequently, the potential sex-dependent action of antidepressants remains elusive. We investigated whether Flinders sensitive line (FSL) of rats, a model of depression, would present sex-differentiated responses to antidepressant treatment. FSL and Sprague—Dawley rats were treated with clomipramine 10 mg/kg/day for 14 days. Subsequently, they were subjected to either a single session of the forced swim test or an estimation of serotonergic function in the prefrontal cortex, hippocampus, amygdala and hypothalamus. Male FSL displayed increased immobility duration, decreased active behaviours, increased serotonin tissue levels and a reduced serotonin turnover rate in most brain areas studied. Female FSL showed a distinct profile, consisting of decreased immobility latency, increased climbing duration, limited serotonergic deviations and no difference in the serotonin turnover rate in comparison with controls. Interestingly, despite baseline differences, clomipramine treatment reversed all relevant behavioural responses and increased the serotonin turnover rate in both sexes. However, the latter effect was remarkably more pronounced in females. It is concluded that, in this animal model of depression, chronic clomipramine treatment attenuated baseline sex differences in the phenotype while maintaining or intensifying the sex differentiation in the serotonergic endophenotype.

Sex differences in oxidant/antioxidant balance under a chronic mild stress regime.

The deterioration of homeostasis between oxidant/antioxidant species may represent an important mechanism linking psychological stress to cardiovascular risk despite the many sex differences in stress responsiveness. The goal of the present study was to investigate the influence of chronic mild stress (CMS), a widely accepted animal model of depression, on oxidative homeostasis-allostasis markers and sICAM-1, a marker of endothelial injury, in the serum of Wistar rats, by taking into account the effect of sex. After six weeks of exposure to mild unpredictable environmental stressors, both male and female rat groups displayed typical changes in hedonic status (anhedonia), which is a core symptom of human depression. Control female rats had higher (nitrite and nitrate) NOx, lower malondealdehyde (MDA) levels with lower activity of antioxidant enzymes and sICAM-1 levels than did control males. CMS induced oxidant/antioxidant responses in both sexes. Females tended to increase their nitric oxide (NO) levels further, while MDA levels did not reach those of males, thus retaining significantly higher NO bioavailability than in males. Concerning the antioxidant enzymes, CMS-females exhibited significantly higher glutathione peroxidase (GPx) activity and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity compared to CMS-males. The CMS response in females was accompanied by lower sICAM-1 levels than in males, suggesting lower endothelial injury. In conclusion, the results of the present study showed that CMS induces different oxidative stress and compensatory responses in both sexes probably due to differences in the mechanisms regulating oxidant/antioxidant pathways.