Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex

Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1–CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females.     

Prenatal nicotine exposure alters postnatal cardiorespiratory integration in young male but not female rats

The present study tested the hypothesis that prenatal nicotine exposure (PNE) induces sex specific alternations in indices of cardiorespiratory coupling during early development. Rat pups exposed to either nicotine (6 mg/kg/day) or saline (control) in utero were chronically instrumented with ECG electrodes for measurement of heart rate (HR) and respiratory frequency (RF) was monitored by whole body plethysmography on postnatal days (P)13, P16 and P26. PNE had no identifiable effect on resting respiratory frequency (RF) in either sex. There was however a strong trend (p = 0.057) for resting HR to be elevated by PNE in male offspring only. Alternatively, the HR response to hypoxia (10% O₂), was significantly blunted at P13 but significantly elevated at P26 s in the absence of any significant change in RF in PNE males only. Indicators of respiratory sinus arrhythmia (RSA) were also significantly reduced in P26 PNE males. No significant effects of PNE on HR, RF or RSA were identified in female offspring at any age. Our results demonstrate that PNE induces very specific changes in cardiorespiratory integration at select postnatal ages and these changes are more prominent in males. Additionally, alternations in cardiorespiratory integration appear to persist into later development in males only, potentially increasing the risk for cardiovascular diseases such as hypertension later in life.     

Maternal deprivation in the early versus late postnatal period differentially affects growth and stress-induced corticosterone responses in adolescent rats

Periodic maternal deprivation (MD) in the early postnatal period leads to permanently altered responsibility of the hypothalamus-pituitary-adrenocortical (HPA) axis to various types of stress. However, no reports appear to have described the effect of periodic MD under different conditions on growth of the developing rat and responsibility of the HPA axis to immobilization stress in adolescent rats. Furthermore, although body weight changes are known to affect stress responsibility, their relationship under periodic MD is not clear. The present study therefore used 4 different types of periodic MD: for 12 h/day from postnatal day (P)1 to P6 (12E group); for 3 h/day from P1 to P6 (3E group); for 12 h/day from P16 to P21 (12L group); and for 3 h/day from P16 to P21 (3L group). Mean body weights were less in the 3E and 12E groups than in the control group until at least 9 weeks old, although body weight gain in the 3L and 12L groups was only transiently affected. Stress-induced corticosterone levels in the 3E and 12E groups did not return to basal levels until at least 330 min after the termination of stress, while temporal variations of stress-induced corticosterone levels did not differ significantly between the 3L, 12L and control groups. Periodic MD in the first postnatal week affected growth of developing rats and responsibility of the HPA axis to immobilization stress in adolescent rats, and the extent of this modification was larger with MD for 12 h/day than with MD for 3 h/day. Conversely, periodic MD from P16 to P21 had little effect. Periodic MD in the postnatal period induces long-term effects on growth and stress responsibility of the HPA axis. Furthermore, a critical age of the pup at the time of MD exists as well as a critical length of MD for inducing these effects.     

Postnatal experiences influence the behavior in adult male and female Fischer and Lewis rats

The postnatal environment with the rat pups’ dam as the most important regulator, plays a central role in determining developmental processes of the offspring. Early disturbances of the dam-pup-dyade, like separation from the dam for hours (maternal deprivation, MD), or a short period of separation, and exposure to novelty, like the handling stimulation (HA), might induce long-lasting changes within the individual. To further investigate the susceptibility to these postnatal manipulations with regard to both, sex and genetic background, we used male and female Fischer (F344) and Lewis (LEW) rats.     

Stress differentially alters mu opioid receptor density and trafficking in parvalbumin‐containing interneurons in the female and male rat hippocampus

Stress differentially affects hippocampal‐dependent learning relevant to addiction and morphology in male and female rats. Mu opioid receptors (MORs), which are located in parvalbumin (PARV)‐containing GABAergic interneurons and are trafficked in response to changes in the hormonal environment, play a critical role in promoting principal cell excitability and long‐term potentiation. Here, we compared the effects of acute and chronic immobilization stress (AIS and CIS) on MOR trafficking in PARV‐containing neurons in the hilus of the dentate gyrus in female and male rats using dual label immunoelectron microscopy. Following AIS, the density of MOR silver‐intensified gold particles (SIGs) in the cytoplasm of PARV‐labeled dendrites was significantly reduced in females (estrus stage). Conversely, AIS significantly increased the proportion of cytoplasmic MOR SIGs in PARV‐labeled dendrites in male rats. CIS significantly reduced the number of PARV‐labeled neurons in the dentate hilus of males but not females. However, MOR/PARV‐labeled dendrites and terminals were significantly smaller in CIS females, but not males, compared with controls. Following CIS, the density of cytoplasmic MOR SIGs increased in PARV‐labeled dendrites and terminals in females. Moreover, the proportion of near‐plasmalemmal MOR SIGs relative to total decreased in large PARV‐labeled dendrites in females. After CIS, no changes in the density or trafficking of MOR SIGs were seen in PARV‐labeled dendrites or terminals in males. These data show that AIS and CIS differentially affect available MOR pools in PARV‐containing interneurons in female and male rats. Furthermore, they suggest that CIS could affect principal cell excitability in a manner that maintains learning processes in females but not males. Synapse 67:757–772, 2013 . © 2013 Wiley Periodicals, Inc.     

Reduced serotonin synthesis during early embryogeny changes effect of subsequent prenatal stress on persistent pain in the formalin test in adult male and female rats

The considerable evidence supporting a role for serotonin (5-HT) in the embryonic formation of CNS, mediation of prenatal stress, and pain processing is reviewed. Long-term influences of prenatal 5-HT depletion as well as its combination with prenatal stress effects on tonic nociceptive system in 90-day-old Wistar rats were studied in the formalin test. Pregnant dams were injected with para-chlorophenylalanine (pCPA, 400 mg/kg/2 ml, ip), producing 5-HT depletion during the early period of fetal serotonergic system development. The adult offspring from pCPA-treated dams revealed changes in behavioral indices of persistent pain (flexing + shaking and licking) in the formalin test (2.5%, 50 microl) that were accompanied by irreversible morphological alterations in the dorsal raphe nuclei. In the other series of experiments, the role of 5-HT in the mediation of prenatal stress on the behavioral indices of persistent pain was investigated in the adult offspring from dams with 5-HT depletion followed by restraint stress. Stress during the last embryonic week caused much more increase in flexing + shaking and licking in the second tonic phase of the response to formalin in offspring from pCPA- than saline-treated (control) dams. The former was characterized by alterations in the durations of the interphase, the second phase, and the whole behavioral response too. In offspring from pCPA-treated dams, sex dimorphism was revealed in tonic pain evaluated by licking. Together with our previous results in juvenile rats demonstrating the necessity of definite level of prenatal 5-HT for normal development of tonic nociceptive system, the present pioneering findings obtained in adult rats indicate that prenatal 5-HT depletion causes long-term morphological abnormalities in the dorsal raphe nuclei accompanied by alterations in behavioral indices of tonic pain. Early prenatal 5-HT depletion increases vulnerability of tonic nociceptive circuits to the following prenatal stress.     

Characterization of spatial performance in male and female Long-Evans rats by means of the Morris water task and the ziggurat task

Sex differences are prominent influences on spatial performance. One of the most common tasks to assess sex differences in spatial navigation in rodents is the Morris water task (MWT). In this task rats swim in a pool of water to locate a hidden platform employing the topographical relationships among the distal visual cues, pool wall, and goal location. Some evidence suggests that male rats display superior performance relative to females in the MWT. It is unknown, however, to what extent the sex difference in rats is task-dependent. This study compared the performance of male and female Long-Evans rats in the wet-land MWT versus the dry-land ziggurat task (ZT). The ZT represents a new dry-land task in which rats explore an arena with 16 ziggurat pyramids to locate food rewards. Several behavioural parameters, including latency, path length, path speed, probe trial performance, errors, and the number of returns were used as indices of spatial learning and memory. While males and females did not display significant differences in the traditional measures of spatial navigation within MWT, they displayed a robust sex difference in all measures of the ZT. These results indicate task-specific sex differences in spatial performance. Our findings suggest that males and females may employ different learning strategies in the MWT and ZT and that the latter task provides a more favourable task for assessing sex differences in rats.     

Microglia react to partner loss in a sex- and brain site-specific manner in prairie voles

Positive social relationships are paramount for the survival of mammals and beneficial for mental and physical health, buffer against stressors, and even promote appropriate immune system functioning. By contrast, impaired social relationships, social isolation, or the loss of a bonded partner lead to aggravated physical and mental health. For example, in humans partner loss is detrimental for the functioning of the immune system and heightens the susceptibility for the development of post-traumatic stress disorders, anxiety disorders, and major depressive disorders. To understand potential underlying mechanisms, the monogamous prairie vole can provide important insights. In the present study, we separated pair bonded male and female prairie voles after five days of co-housing, subjected them to the forced swim test on the fourth day following separation, and studied their microglia morphology and activation in specific brain regions. Partner loss increased passive stress-coping in male, but not female, prairie voles. Moreover, partner loss was associated with microglial priming within the parvocellular region of the paraventricular nucleus of the hypothalamus (PVN) in male prairie voles, whereas in female prairie voles the morphological activation within the whole PVN and the prelimbic cortex (PrL) was decreased, marked by a shift towards ramified microglial morphology. Expression of the immediate early protein c-Fos following partner loss was changed within the PrL of male, but not female, prairie voles. However, the loss of a partner did not affect the investigated aspects of the peripheral immune response. These data suggest a potential sex-dependent mechanism for the regulation of microglial activity following the loss of a partner, which might contribute to the observed differences in passive stress-coping. This study furthers our understanding of the effects of partner loss and its short-term impact on the CNS as well as the CNS immune system and the peripheral innate immune system in both male and female prairie voles.     

Maternal separation alters ICSS responding in adult male and female rats, but morphine and naltrexone have little affect on that behavior

Prior research has provided evidence that the early postnatal environment can have long lasting effects on both the physiology and behavior of offspring. This is modeled in rats by using a maternal separation paradigm in which pups are separated from their mother for a few hours daily during their first two postnatal weeks. While this model has been used extensively to study stress effects and anxiety, less research has been done to examine how these separations affect measures of reward and reinforcement in adulthood. The current study investigated the impact of maternal separation (MS) on intracranial self-stimulation (ICSS) maintained responding in male and female offspring, and the effects of morphine (0.3-3.0 mg/kg) and naltrexone (0.1-10 mg/kg) on that responding. Rearing condition (MS or non-handled, NH) significantly altered response rates during acquisition in both sexes, with NH offspring exhibiting the highest rates. Group differences in baseline responding on a progressive ratio (PR-2) schedule of reinforcement were evident only in females, with MS females having response rates 50% lower than NH females. Neither morphine nor naltrexone differentially affected either rearing group. Sex impacted NH offspring: males acquired responding more readily, but females had higher response rates and breakpoints during all other phases of the experiment. In MS offspring, no sex differences were observed during acquisition, but during all other phases males had higher response rates and breakpoints than females. These results indicate that maternal separation during the first two postnatal weeks can have long-term effects on responding for ICSS, but these effects do not appear tied to endogenous opioid systems in the lateral hypothalamus.     

Early maternal deprivation in rats induces gender-dependent effects on developing hippocampal and cerebellar cells

Adult animals submitted to a single prolonged episode of maternal deprivation [24 h, postnatal day 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. According to the neurodevelopmental theory, these behavioral deficits might be mediated by detrimental neurodevelopmental processes that might be associated, at least partially, with stress-induced corticosterone responses. In order to address this hypothesis, we have focused on the hippocampus and cerebellar cortex, two brain regions that show high density of glucocorticoid receptors, and analyzed possible neuronal and glial alterations by immunohistochemical techniques. To evaluate the presence of degenerated neurons we used Fluoro-Jade-C (FJ-C) staining and for the study of astrocytes we employed glial fibrillary acidic protein (GFAP). Within control animals, females showed significantly more GFAP positive cells than males and a trend towards more FJ-C positive cells. Maternal deprivation induced neuronal degeneration and astroglial changes in the hippocampus and cerebellar cortex of neonatal rats that, in general, were more marked in males. This differential effect may be attributable to a greater vulnerability of males to this kind of early environmental insult and/or to sex-dependent differences in the onset and/or progression of the effects. The present experimental procedure may be instrumental in elucidating sex-dependent mechanisms of neurodevelopmental psychiatric disorders with a basis in early environmental insults.     

Prenatal exposure to sodium valproate alters androgen receptor expression in the developing cerebellum in a region and age specific manner in male and female rats

Valproic acid (VPA) is an anti-epileptic drug with teratogenicity activity that has been related to autism. In rodents, exposure to VPA in utero leads to brain abnormalities similar than those reported in the autistic brain. Particularly, VPA reduces the number of Purkinje neurons in the rat cerebellum parallel to cerebellar abnormalities found in autism. Thus, we injected pregnant females on embryonic day 12 either with VPA (600 mg/kg, i.p.) or 0.9% saline solution and obtained the cerebellum from their offspring at different postnatal time points. Testosterone has been linked to autism and plays an important role during brain development. Therefore, we identified and analyzed the androgen receptor (AR) by immunohistochemistry and densitometry, respectively. We found VPA decreases AR density in the superficial Purkinje layer only in cerebellar lobule 8 at PN7, but increased it at PN14 compared to control in males. In females, VPA decreased AR density in the superficial Purkinje layer in cerebellar lobule 6 at PN14, but increased it in lobule 9 at the same time point. No differences were found in the deep Purkinje layer of any cerebellar lobule in terms of AR density neither in males nor females. We additionally found a particular AR density decreasing in both superficial and deep regions across development in the majority of cerebellar lobules in males, but in all cerebellar lobules in females. Thus, our results indicate that VPA disrupts the AR ontogeny in the developing cerebellum in an age and region specific manner in male and female rats. Future epigenetic studies including the evaluation of histone deacetylases (HDAC’s) might shed light these results as HDAC’s are expressed by Purkinje neurons, interact with the AR and are VPA targets. This work contributes to the understanding of the cerebellar development and it might help to understand the role of the cerebellum in neurodevelopmental disorders such as autism.     

Effects of a single footshock followed by situational reminders on HPA axis and behaviour in the aversive context in male and female rats

Gender is an important factor in the vulnerability to develop psychopathologies. At the biological level, stress-related pathologies such as depression or post-traumatic stress disorder (PTSD) are associated with profound disturbances of the hypothalamo-pituitary-adrenal (HPA) axis. The aim of the present study was to assess sex-differences in the long-term effect of an intense stressful procedure on HPA function and behaviour in the aversive context in rats. Female and male rats experienced an aversive procedure consisting in an electric footshock (2mA, 10s) in a dark chamber followed by 3 weekly situational reminders (SR, 2min in the white chamber close to the footshock chamber). Our results indicate that 41 days after the end of the aversive procedure, female rats showed an increase of the corticosterone negative feedback in response to restraint stress, whereas such effect was not observed in males. Despite this change in the hormonal response, glucocorticoid receptors mRNA expression in the hippocampus was not affected in shocked females. In contrast, a significant increase of the mineralocorticoid receptors mRNA was observed in the CA2 of the hippocampus in shocked males. Finally, CRH mRNA levels in the paraventricular nucleus of the hypothalamus (PVN) were decreased in both female and male animals exposed to the aversive procedure. Behavioural observation revealed that shocked males and shocked females showed a high level of avoidance. However, the latency to visit the shock box was lower in females, which spent also more time in this area than males. In conclusion, our results suggest that gender might be a key factor impacting the direction of the effects induced by an intense stress. Interestingly, only females exhibited an increased negative feedback of the HPA axis response to stress, which could parallel endocrine changes of PTSD.     

Epigenetic programming of the stress response in male and female rats by prenatal restraint stress

Exposure to hostile conditions results in a series of coordinated responses aimed at enhancing the probability of survival. The activation of the hypothalamo-pituitary-adrenocortical (HPA) axis plays a pivotal role in the stress response. While the short-term activation of the HPA axis allows adaptive responses to the challenge, in the long run this can be devastating for the organism. In particular, life events occurring during the perinatal period have strong long-term effects on the behavioral and neuroendocrine response to stressors. In male and female rats exposed to prenatal restraint stress (PRS), these effects include a long-lasting hyperactivation of the HPA response associated with an altered circadian rhythm of corticosterone secretion. Furthermore, male animals exhibit sleep disturbances. In males, these HPA dysfunctions have been reported in infant, young, adult and aged animals, thus suggesting a permanent effect of early stress. Interestingly, after exposure to an intense inescapable footshock, female PRS rats durably exhibit a blunted corticosterone secretion response to stress. In male PRS rats exposed to an alcohol challenge, the HPA axis is similarly hyporesponsive. Rats exposed to PRS also show behavioral disturbances. Both male and female PRS rats show high anxiety levels and depression-like behavior during adulthood, although some studies suggest that female PRS rats present low anxiety levels. With ageing, male and female PRS rats exhibit memory impairments in hippocampus-dependent tasks, while female PRS rats improve their memory performance during adulthood. The gender effect on behavior seems to be related to a reduction in hippocampal plasticity in male PRS rats, and an increase in female PRS rats. Despite the permanent imprinting induced by early stress, the dysfunctions observed after PRS can be reversed by environmental or pharmacological strategies such as environmental enrichment or antidepressive and neurotrophic treatments. Mechanisms underlying the effects of PRS on the offspring remain largely unknown. However, previous studies have demonstrated that maternal glucocorticoids during pregnancy play an important role in the HPA disturbances reported in male offspring. Finally, gestational stress has long-lasting effects on the HPA axis and on behavior in the dams. Alterations in maternal behavior could thus also make a strong contribution to the long-term effects of PRS, through epigenetic mechanisms.     

Sex differences in the association between stressor-evoked interleukin-6 reactivity and C-reactive protein

Individuals differ consistently in the magnitude of their inflammatory responses to acute stressors, with females often showing larger responses than males. While the clinical significance of these individual differences remains unclear, it may be that greater inflammatory responses relate to increased systemic inflammation and thereby risk for chronic inflammatory disease. Here, we examined whether acute stressor-evoked interleukin (IL)-6 responses associate with resting levels of C-reactive protein (CRP), a marker of systemic inflammation, and whether this association differs by sex. Subjects were 57 healthy midlife adults (30–51 years; 33% female; 68% white). Blood was drawn before and 30-min after two mental stress tasks: a multisource interference task and a Stroop color word task. Hierarchical regressions controlling for age, sex, race, and BMI tested whether stressor-evoked IL-6 responses were associated with resting CRP and whether this association differed by sex. Results indicated that sex and stressor-evoked IL-6 responses interacted to predict CRP (ΔR² = 0.08, B = −1.33, β = −0.39, p = 0.02). In males, larger stressor-evoked IL-6 responses associated with higher CRP, whereas in females, stressor-evoked IL-6 responses showed a non-significant negative association with CRP. These findings indicate that inflammatory responses to acute stressors associate with resting levels of CRP; however, this association differs by sex. Previous literature suggests that there are sex differences in stressor-evoked IL-6 responses, but this is the first study to show sex differences in the relationship between acute inflammatory responses and systemic inflammation. The contribution of these sex differences to inflammatory disease risk warrants further investigation.