High-fat diet and aging-associated memory impairments persist in the absence of microglia in female rats.

Aging is associated with a priming of microglia such that they are hypersensitive to further immune challenges. As such high-fat diet during aging can have detrimental effects on cognition that is not seen in the young. However, conflicting findings also suggest that obesity may protect against cognitive decline during aging. Given this uncertainty we aimed here to examine the role of microglia in high-fat, high-sucrose diet (HFSD)-induced changes in cognitive performance in the aging brain. We hypothesised that 8 weeks of HFSD-feeding would alter microglia and the inflammatory milieu in aging and worsen aging-related cognitive deficits in a microglia-dependent manner. We found that both aging and HFSD reduced hippocampal neuron numbers and open field exploration; they also impaired recognition memory. However, the aging-related deficits occurred in the absence of a pro-inflammatory response and the deficits in memory performance persisted after depletion of microglia in the Cx3cr1-Dtr knock-in rat. Our data suggest that mechanisms additional to the acute microglial contribution play a role in aging- and HFSD-associated memory dysfunction.

Long-term role of neonatal microglia and monocytes in ovarian health.

Early life microglia are essential for brain development, and developmental disruption in microglial activity may have long-term implications for the neuroendocrine control of reproduction. We and others have previously shown that early life immune activation compromises the long-term potential for reproductive function in females. However, the supportive role of microglia in female reproductive development is still unknown. Here, we examined the long-term programming effects of transient neonatal microglial and monocyte ablation on hypothalamic-pituitary-gonadal (HPG) axis function in female rats. We employed a Cx3cr1-Dtr transgenic Wistar rat model to acutely ablate microglia and monocytes, commencing on either postnatal day (P) 7 or 14, since the development of the HPG axis in female rodents primarily occurs during the first two to three postnatal weeks. After an acutely diminished expression of microglia and monocyte genes in the brain and ovaries, respectively, microglia had repopulated the brain by P21, albeit that cellular complexity was still reduced in both groups at this time. Removal of microglia and monocytes on P7, but not P14 reduced circulating luteinising hormone levels in adulthood and ovarian gonadotropin receptors mRNA. These changes were notably associated with fewer primary and antral follicles in these rats. These data suggest that transient ablation of microglia and monocytes at the start of the second but not the third postnatal week has long-term effects on ovarian health. The findings highlight the important developmental role of a healthy immune system for female potential reproductive capacity and the importance of critical developmental periods to adult ovarian health.

Maternal diet before and during pregnancy modulates microglial activation and neurogenesis in the postpartum rat brain.

The implications of poor maternal diet on offspring metabolic and neuroimmune development are well established. Increasing evidence now suggests that maternal obesity and poor diet can also increase the risk of postpartum mood disorders, but the mechanisms are unknown. Here we investigated the effects of a poor, high-fat-high-sugar diet (HFSD) on peripheral and central inflammation, neurogenesis and postpartum anxiety-like behaviours. We hypothesised that long-term consumption of a HFSD pre- and post-conception would increase the levels of circulating cytokines and induce microglial activation, particularly in the arcuate nucleus of the hypothalamus (ARC), as the primary brain region involved in the integration of satiety signalling; and this would lead to increased anxiety, stress responsivity and disrupted neurogenesis. We further hypothesised that these effects would be ameliorated by consumption of a healthier diet during pregnancy - specifically a diet high in omega-3 polyunsaturated fatty acids (PUFAs). As expected, the HFSD significantly increased pre-conception body weight, elevated circulating cytokines and activated microglia in the ARC, as well as in the basolateral amygdala. The HFSD also significantly increased the numbers of immature (doublecortin (DCX)-positive) neurons in the subgranular/granular region of the hippocampus, a neurogenic response that was, surprisingly, mimicked by consumption of a diet high in omega-3 PUFAs. Despite these effects of peri-pregnancy dietary imbalance, we detected no differences in anxiety-like behaviours or hypothalamic-pituitary-adrenal (HPA) axis reactivity between the groups. A shift to a healthier diet post-conception reversed the peripheral inflammation and alleviated the microglial activation. These novel data indicate the importance of a balanced peri-pregnancy diet and highlight the need for future research into key triggers that alter the neuroimmune balance in the maternal brain.

The role of microglia in the second and third postnatal weeks of life in rat hippocampal development and memory.

Microglia are resident immune cells of the central nervous system (CNS). In adulthood they are involved in surveillance and responses to pathogens and injury and prenatally they play a role in brain development. However, the role of microglia during the early postnatal period and how they impact development long-term remains poorly understood. Here, to investigate the specific role of microglia in postnatal development, we used a Cx3cr1-Dtr transgenic Wistar rat model to acutely ablate microglia from either postnatal day (P) 7 or 14. We specifically assessed how transient microglial ablation affected astrocytes and neurons acutely, during the juvenile period, and in adulthood. Hippocampal microglial numbers remained low at P21 in the P7-ablated animals and complexity remained reduced after P14-ablation. This protracted effect on these key immune cells led to a small but significant increase in CA1 mature neuron numbers and a significant increase in astrocyte density in the subgranular dentate gyrus in adults that had their microglia ablated at P14. However, these histological differences were small, and spatial and recognition memory in novel objection and place recognition tests were not affected. Overall, our data reveal for the first time that the transient depletion of microglia during the neonatal period impacts briefly on the brain but that the long-lasting effects are minimal. Neonatal microglia may be dispensable in the establishment of hippocampal brain function. These data also imply that novel therapeutic anti-inflammatories that cross the blood-brain barrier to inhibit microglia are unlikely to have long-term negative consequences if administered in the neonatal period.

Consequences of early life overfeeding for microglia - Perspectives from rodent models.

The early life period is crucially important to how the individual develops, and environmental and lifestyle challenges during this time can lead to lasting programming effects on the brain and immune system. In particular, poor diet in early development can lead to long-term negative metabolic and cognitive outcomes, with those who over-eat in early development being at risk of obesity and poor learning and memory throughout their adult lives. Current research has identified a neuroinflammatory component to this metabolic and cognitive programming that can potentially be manipulated to restore a healthy phenotype. Thus, early life over-feeding in a rat model leads to microglial priming and an exacerbated microglial response to immune challenge when the rats reach adulthood. Microglial responses to a learning task are also impaired. To specifically investigate the role of microglia in these programming effects our group has developed a novel transgenic rat with a diphtheria toxin receptor insertion in the promoter region for the Cx3cr1 gene, expressed on microglia and monocytes; allowing us to conditionally ablate microglia throughout the brain. With this model we reveal that microglia have a direct role in regulating feeding behavior and modifying cognition, but are not likely to be the sole mechanism by which early life overfeeding confers lasting neuroimmune and cognitive effects. Additional work implicates changes to the hypothalamic-pituitary-adrenal axis in this. Together these data highlight the importance of dietary choices in early life and the potential for positive interventions targeting the neuroimmune and neuroendocrine stress systems to reverse such programming damage.

Glial remodeling enhances short-term memory performance in Wistar rats.

BACKGROUND: Microglia play a key role in neuronal circuit and synaptic maturation in the developing brain. In the healthy adult, however, their role is less clear: microglial hyperactivation in adults can be detrimental to memory due to excessive synaptic pruning, yet learning and memory can also be impaired in the absence of these cells. In this study, we therefore aimed to determine how microglia contribute to short-term memory in healthy adults. METHODS: To this end, we developed a Cx3cr1-Dtr transgenic Wistar rat with a diphtheria toxin receptor (Dtr) gene inserted into the fractalkine receptor (Cx3cr1) promoter, expressed on microglia and monocytes. This model allows acute microglial and monocyte ablation upon application of diphtheria toxin, enabling us to directly assess microglia's role in memory. RESULTS: Here, we show that short-term memory in the novel object and place recognition tasks is entirely unaffected by acute microglial ablation. However, when microglia repopulate the brain after depletion, learning and memory performance in these tasks is improved. This transitory memory enhancement is associated with an ameboid morphology in the newly repopulated microglial cells and increased astrocyte density that are linked with a higher density of mature hippocampal synaptic spines and differences in pre- and post-synaptic markers. CONCLUSIONS: These data indicate that glia play a complex role in the healthy adult animal in supporting appropriate learning and memory and that subtle changes to the function of these cells may strategically enhance memory.

Obesity after neonatal overfeeding is independent of hypothalamic microgliosis.

The early-life environment is important in programming brain development, and metabolic disruptions at this time can have long-lasting effects. Previously, we have shown that rats overfed for the first 3 weeks of their neonatal life maintain obesity into adulthood. Neonatal overfeeding also leads to primed hypothalamic and hippocampal microglia that are hyper-responsive to an immune challenge in adulthood. However, whether this microglial priming contributes to the obese phenotype and whether it is possible to reverse either the obesity or the microglial priming are not clear. In the present study, we hypothesised that an intervention with minocycline during the juvenile period (postnatal day 21-42) would normalise both the microglial priming and obesity. To induce obesity in neonatal Wistar rats, we manipulated the litter sizes in which they were suckled, yielding litters of 12 (control-fed) or four (neonatally overfed). After weaning, we administered minocycline i.p. every second day for a 3-week period and examined body composition and microglial profiles 24 hours following an immune challenge with lipopolysaccharide. As demonstrated previously, neonatal overfeeding resulted in prolonged weight gain. However, minocycline failed to reverse this effect. Minocycline did reverse microglial priming in feeding-related regions of the hypothalamus, with minimal effects on pro-inflammatory cytokines and on microglial number and morphology in the hippocampus. Thus, the programming effect of neonatal overfeeding on microglial priming can be ameliorated by minocycline later in life. However, the persistent obesity seen after neonatal overfeeding is likely not driven by changes in hypothalamic inflammation and microglial activity.

Chronic predator stress in female mice reduces primordial follicle numbers: implications for the role of ghrelin.

Chronic stress is a known suppressor of female reproductive function. However, attempts to isolate single causal links between stress and reproductive dysfunction have not yet been successful due to their multi-faceted aetiologies. The gut-derived hormone ghrelin regulates stress and reproductive function and may therefore be pivotal in the neuroendocrine integration of the hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes. Here, we hypothesised that chronic stress disrupts ovarian follicle maturation and that this effect is mediated by a stress-induced increase in acyl ghrelin and activation of the growth hormone secretatogue receptor (GHSR). We gave C57BL/6J female mice 30 min daily chronic predator stress for 4 weeks, or no stress, and gave them daily GHSR antagonist (d-Lys3-GHRP-6) or saline. Exposure to chronic predator stress reduced circulating corticosterone, elevated acyl ghrelin levels and led to significantly depleted primordial follicle numbers. GHSR antagonism stress-dependently altered the expression of genes regulating ovarian responsiveness to gonadotropins and was able to attenuate the stress-induced depletion of primordial follicles. These findings suggest that chronic stress-induced elevations of acyl ghrelin may be detrimental for ovarian follicle maturation.

High-fat diet worsens the impact of aging on microglial function and morphology in a region-specific manner.

Hippocampal microglia are vulnerable to the effects of aging, displaying a primed phenotype and hyper-responsiveness to various stimuli. We have previously shown that short-term high-fat diet (HFD) significantly impairs hippocampal- and amygdala-based cognitive function in the aged without affecting it in the young. Here, we assessed if morphological and functional changes in microglia might be responsible for this. We analyzed hippocampus and amygdala from young and aging rats that had been given three days HFD, a treatment sufficient to cause both hippocampal- and amygdala-dependent cognitive and neuroinflammatory differences in the aged. Aging led to the expected priming of hippocampal microglia in that it increased microglial numbers and reduced branching in this region. Aging also increased microglial phagocytosis of microbeads in the hippocampus, but the only effect of HFD in this region was to increase the presence of enlarged synaptophysin boutons in the aged, indicative of neurodegeneration. In the amygdala, HFD exacerbated the effects of aging on microglial priming (morphology) and markedly suppressed phagocytosis without notably affecting synaptophysin. These data reveal that, like the hippocampus, the amygdala displays aging-related microglial priming. However, the microglia in this region are also uniquely vulnerable to the detrimental effects of short-term HFD in aging.

Conditional microglial depletion in rats leads to reversible anorexia and weight loss by disrupting gustatory circuitry.

Microglia are highly sensitive to dietary influence, becoming activated acutely and long-term by high fat diet. However, their role in regulating satiety and feeding in healthy individuals remains unclear. Here we show that microglia are essential for the normal regulation of satiety and metabolism in rats. Short-term microglial depletion in a Cx3cr1-Dtr rat led to a dramatic weight loss that was largely accounted for by an acute reduction in food intake. This weight loss and anorexia were not likely due to a sickness response since the rats did not display peripheral or central inflammation, withdrawal, anxiety-like behavior, or nausea-associated pica. Hormonal and hypothalamic anatomical changes were largely compensatory to the suppressed food intake, which occurred in association with disruption of the gustatory circuitry at the paraventricular nucleus of the thalamus. Thus, microglia are important in supporting normal feeding behaviors and weight, and regulating preference for palatable food. Inhibiting this circuitry is able to over-ride strong compensatory drives to eat, providing a potential target for satiety control.

High-fat diet and aging interact to produce neuroinflammation and impair hippocampal- and amygdalar-dependent memory.

More Americans are consuming diets higher in saturated fats and refined sugars than ever before, and based on increasing obesity rates, this is a growing trend among older adults as well. While high saturated fat diet (HFD) consumption has been shown to sensitize the inflammatory response to a subsequent immune challenge in young adult rats, the inflammatory effect of HFD in the already-vulnerable aging brain has not yet been assessed. Here, we explored whether short-term (3 days) consumption of HFD would serve as a neuroinflammatory trigger in aging animals, leading to cognitive deficits. HFD impaired long-term contextual (hippocampal dependent) and auditory-cued fear (amygdalar dependent) memory in aged, but not young adult rats. Short-term memory performance for both tasks was intact, suggesting that HFD impairs memory consolidation processes. Microglial markers of activation Iba1 and cd11b were only increased in the aged rats, while MHCII was further amplified by HFD. Furthermore, these HFD-induced long-term memory impairments were accompanied by IL-1ß protein increases in both the hippocampus and amygdala in aged rats. Central administration of IL-1RA in aged rats following conditioning mitigated both contextual and auditory-cued fear memory impairments caused by HFD, strongly suggesting that IL-1ß plays a critical role in these effects. Voluntary wheel running, known to have anti-inflammatory effects in the hippocampus, rescued hippocampal-dependent but not amygdalar-dependent memory impairments caused by HFD. Together, these data suggest that short-term consumption of HFD can lead to memory deficits and significant brain inflammation in the aged animal, and strongly suggest that appropriate diet is crucial for cognitive health.

Effects of exercise on adolescent and adult hypothalamic and hippocampal neuroinflammation.

Adolescence is a period of significant brain plasticity that can be affected by environmental factors, including the degree of physical activity. Here we hypothesized that adolescent rats would be more sensitive to the beneficial metabolic and anti-inflammatory effects of voluntary exercise than adult rats, whose more mature brains have less capacity for plasticity. We tested this by giving adolescent and adult Wistar rats four weeks' voluntary access to running wheels. At the end of this period we assessed metabolic effects, including weight and circulating leptin and ghrelin, as well as performance in a novel object recognition test of memory and central changes in neuronal proliferation, survival, synaptic density, and inflammatory markers in hippocampus. We found exercise reduced fat mass and circulating leptin levels in both adults and adolescents but suppressed total weight gain and lean mass in adults only. Exercise stimulated neuronal proliferation in the suprapyramidal blade of the dentate gyrus in both adults and adolescents without altering the number of mature neurons during this time frame. Exercise also increased dentate microglial numbers in adolescents alone and microglial numbers in this region were inversely correlated with performance in the novel object recognition test. Together these data suggest that adolescent hippocampal microglia are more sensitive to the effects of exercise than those of adults, but this leads to no apparent improvement in recognition memory. © 2016 Wiley Periodicals, Inc.

Neonatal overfeeding induces early decline of the ovarian reserve: Implications for the role of leptin.

Early life nutrition is crucial for reproduction. Overweight and obese girls are more likely to experience early menarche, increasing the risk of adult disease. We have previously demonstrated neonatal overfeeding in the rat leads to accelerated growth, early puberty and increased circulating levels of leptin, an adipocyte-derived hormone that regulates puberty. However, the long-term consequences of accelerated puberty and metabolic dysfunction on ovarian reserve are unknown. Here we show that neonatal overfeeding reduced the number of ovarian follicles in adult rats; specifically, the primordial follicle pool was reduced compared to controls. The reduction of ovarian reserve coincided with a diminished release of pituitary gonadotropins at ovulation and altered expression of ovarian markers important for follicular recruitment and survival. These changes were associated with increased levels of ovarian leptin and its receptor. Postnatal administration of leptin antagonist did not reverse the weight gain induced by early life overfeeding, but rescued the decline in the primordial follicle pool and abolished the differences in circulating leptin and gonadotropins. Our findings suggest that the acute effects of elevated circulating leptin may be responsible for the long-term reproductive outcomes after neonatal overfeeding, leading to premature ovarian ageing and changes in reproductive efficiency.

Overfeeding during a critical postnatal period exacerbates hypothalamic-pituitary-adrenal axis responses to immune challenge: a role for adrenal melanocortin 2 receptors.

Early life diet can critically program hypothalamic-pituitary-adrenal (HPA) axis function. We have previously shown rats that are overfed as neonates have exacerbated pro-inflammatory responses to immune challenge with lipopolysaccharide (LPS), in part by altering HPA axis responses, but how this occurs is unknown. Here we examined neonatal overfeeding-induced changes in gene expression in each step of the HPA axis. We saw no differences in glucocorticoid or mineralocorticoid receptor expression in key regions responsible for glucocorticoid negative feedback to the brain and no differences in expression of key HPA axis regulatory genes in the paraventricular nucleus of the hypothalamus or pituitary. On the other hand, expression of the adrenal melanocortin 2 receptor (MC2R) is elevated after LPS in control rats, but significantly less so in the neonatally overfed. The in vitro adrenal response to ACTH is also dampened in these rats, while the in vivo response to ACTH does not resolve as efficiently as it does in controls. These data suggest neonatal diet affects the efficiency of the adrenally-mediated response to LPS, potentially influencing how neonatally overfed rats combat bacterial infection.

Neonatal overfeeding attenuates acute central pro-inflammatory effects of short-term high fat diet.

Neonatal obesity predisposes individuals to obesity throughout life. In rats, neonatal overfeeding also leads to early accelerated weight gain that persists into adulthood. The phenotype is associated with dysfunction in a number of systems including paraventricular nucleus of the hypothalamus (PVN) responses to psychological and immune stressors. However, in many cases weight gain in neonatally overfed rats stabilizes in early adulthood so the animal does not become more obese as it ages. Here we examined if neonatal overfeeding by suckling rats in small litters predisposes them to exacerbated metabolic and central inflammatory disturbances if they are also given a high fat diet in later life. In adulthood we gave the rats normal chow, 3 days, or 3 weeks high fat diet (45% kcal from fat) and measured peripheral indices of metabolic disturbance. We also investigated hypothalamic microglial changes, as an index of central inflammation, as well as PVN responses to lipopolysaccharide (LPS). Surprisingly, neonatal overfeeding did not predispose rats to the metabolic effects of a high fat diet. Weight changes and glucose metabolism were unaffected by the early life experience. However, short term (3 day) high fat diet was associated with more microglia in the hypothalamus and a markedly exacerbated PVN response to LPS in control rats; effects not seen in the neonatally overfed. Our findings indicate neonatally overfed animals are not more susceptible to the adverse metabolic effects of a short-term high fat diet but may be less able to respond to the central effects.