The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents.

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1-2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin.

Apold1 deficiency associates with increased arterial thrombosis in vivo.

BACKGROUND: Endothelial cells regulate the formation of blood clots; thus, genes selectively expressed in these cells could primarily determine thrombus formation. Apold1 (apolipoprotein L domain containing 1) is a gene expressed by endothelial cells; whether Apold1 directly contributes to arterial thrombosis has not yet been investigated. Here, we assessed the effect of Apold1 deletion on arterial thrombus formation using an in vivo model of carotid thrombosis induced by photochemical injury. MATERIAL AND METHODS: Apold1 knockout (Apold1-/- ) mice and wild-type (WT) littermates underwent carotid thrombosis induced by photochemical injury, and time to occlusion was recorded. Tissue factor (TF) activity and activation of mitogen-activated protein kinases (MAPKs) and phosphatidyl-inositol-3 kinase (PI3K)/Akt pathways were analysed by colorimetric assay and Western blotting in both Apold1-/- and WT mice. Finally, platelet reactivity was assessed using light transmission aggregometry. RESULTS: After photochemical injury, Apold1-/- mice exhibited shorter time to occlusion as compared to WT mice. Moreover, TF activity was increased in carotid arteries of Apold1-/- when compared to WT mice. Underlying mechanistic markers such as TF mRNA and MAPKs activation were unaffected in Apold1-/- mice. In contrast, phosphorylation of Akt was reduced in Apold1-/- as compared to WT mice. Additionally, Apold1-/- mice displayed increased platelet reactivity to stimulation with collagen compared with WT animals. CONCLUSIONS: Deficiency of Apold1 results in a prothrombotic phenotype, accompanied by increased vascular TF activity, decreased PI3K/Akt activation and increased platelet reactivity. These findings suggest Apold1 as an interesting new therapeutic target in the context of arterial thrombosis.

Rapid Reconfiguration of the Functional Connectome after Chemogenetic Locus Coeruleus Activation.

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain and regulates many fundamental brain functions. Studies in humans have suggested that strong LC activation might shift network connectivity to favor salience processing. To causally test this hypothesis, we use a mouse model to study the effect of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term "chemo-connectomics." We show that LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. Functional connectivity changes strongly correlate with transcript levels of alpha-1 and beta-1 adrenergic receptors across the brain, and functional network connectivity correlates with NE turnover within select brain regions. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection.

Interplay between TETs and microRNAs in the adult brain for memory formation.

5-hydroxymethylation (5-hmC) is an epigenetic modification on DNA that results from the conversion of 5-methylcytosine by Ten-Eleven Translocation (TET) proteins. 5-hmC is widely present in the brain and is subjected to dynamic regulation during development and upon neuronal activity. It was recently shown to be involved in memory processes but currently, little is known about how it is controlled in the brain during memory formation. Here, we show that Tet3 is selectively up-regulated by activity in hippocampal neurons in vitro, and after formation of fear memory in the hippocampus. This is accompanied by a decrease in miR-29b expression that, through complementary sequences, regulates the level of Tet3 by preferential binding to its 3'UTR. We newly reveal that SAM68, a nuclear RNA-binding protein known to regulate splicing, acts upstream of miR-29 by modulating its biogenesis. Together, these findings identify novel players in the adult brain necessary for the regulation of 5-hmC during memory formation.

Probing the germline-dependence of epigenetic inheritance using artificial insemination in mice.

We developed a simple, noninvasive artificial insemination technique to study epigenetic germline inheritance in mice. This technique avoids interfering factors introduced by superovulation, surgery, in vitro culture or mating that can confound the transmission of acquired epigenetic information through the germline. Using a stress model, we demonstrate that our method is suited to test the causal involvement of the male germline in transmitting acquired information from father to offspring.

Hippocampal gene expression induced by cold swim stress depends on sex and handling.

Stress-related disorders such as PTSD and depression are more prevalent in women than men. One reason for such discordance may be that brain regions involved in stress responses are more sensitive to stress in females. Here, we compared the effects of acute stress on gene transcription in the hippocampus of female and male mice, and also examined the involvement of two key stress-related hormones, corticosterone and corticotropin releasing hormone (Crh). Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), we measured gene expression of Fos, Per1 and Sgk1 45 min after exposure to brief cold swim stress. Stress induced a stronger increase in Fos and Per1 expression in females than males. The handling control procedure increased Fos in both sexes, but occluded the effects of stress in males. Further, handling increased Per1 only in males. Sgk1 was insensitive to handling, and increased in response to stress similarly in males and females. The transcriptional changes observed after swim stress were not mimicked by corticosterone injections, and the stress-induced increase in Fos, Per1 and Sgk1 could neither be prevented by pharmacologically blocking glucocorticoid receptor (GR) nor by blocking Crh receptor 1 (Crhr1) before stress exposure. Finally, we demonstrate that the effects are stressor-specific, as the expression of target genes could not be increased by brief restraint stress in either sex. In summary, we find strong effects of acute swim stress on hippocampal gene expression, complex interactions between handling and sex, and a remarkably unique response pattern for each gene. Overall, females respond to a cold swim challenge with stronger hippocampal gene transcription than males, independent of two classic mediators of the stress response, corticosterone and Crh. These findings may have important implications for understanding the higher vulnerability of women to certain stress-related neuropsychiatric diseases.

Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice.

Small non-coding RNAs (sncRNAs) are potential vectors at the interface between genes and environment. We found that traumatic stress in early life altered mouse microRNA (miRNA) expression, and behavioral and metabolic responses in the progeny. Injection of sperm RNAs from traumatized males into fertilized wild-type oocytes reproduced the behavioral and metabolic alterations in the resulting offspring.

Epigenetic inheritance of disease and disease risk.

Epigenetic marks in an organism can be altered by environmental factors throughout life. Although changes in the epigenetic code can be positive, some are associated with severe diseases, in particular, cancer and neuropsychiatric disorders. Recent evidence has indicated that certain epigenetic marks can be inherited, and reshape developmental and cellular features over generations. This review examines the challenging possibility that epigenetic changes induced by environmental factors can contribute to some of the inheritance of disease and disease risk. This concept has immense implications for the understanding of biological functions and disease etiology, and provides potential novel strategies for diagnosis and treatment. Examples of epigenetic inheritance relevant to human disease, such as the detrimental effects of traumatic stress or drug/toxic exposure on brain functions, are reviewed. Different possible routes of transmission of epigenetic information involving the germline or germline-independent transfer are discussed, and different mechanisms for the maintenance and transmission of epigenetic information like chromatin remodeling and small noncoding RNAs are considered. Future research directions and remaining major challenges in this field are also outlined. Finally, the adaptive value of epigenetic inheritance, and the cost and benefit of allowing acquired epigenetic marks to persist across generations is critically evaluated.

The critical period hypothesis of estrogen effects on cognition: Insights from basic research.

BACKGROUND: In addition to its primary role in reproduction estrogen impacts brain areas important for cognition, including the hippocampus and prefrontal cortex. It has been hypothesized that decline in estrogen levels in women following menopause is associated with, or can exacerbate, age-related cognitive decline. However, clinical evidence to support a role for estrogen in preventing cognitive decline in women as they age is equivocal. The critical period hypothesis of estrogen effects on cognition, which proposes that estrogen administration has to be initiated within a critical time period following the loss of ovarian function in order for it to exert positive effects on the central nervous system, is offered as one explanation for inconsistencies across studies. SCOPE OF REVIEW: This review details results from basic research using rodent models investigating the effects of estrogen on cognition in the aging female. Emphasis is placed on work investigating effects of timing of initiation of estrogen administration on its subsequent efficacy. MAJOR CONCLUSIONS: Results of basic research provide support for the critical period hypothesis. Furthermore, results of work in rodent models suggest mechanisms by which the response to estrogen is altered if treatment is initiated following long-term ovarian hormone deprivation. GENERAL SIGNIFICANCE: Understanding if and under what conditions hormone administration following the loss of ovarian function positively affects the brain and behavior could have important implications with regard to female cognitive aging. Results of basic research can contribute to this understanding and provide insight into the complex mechanisms by which estrogen affects cognition.

Transient estradiol exposure during middle age in ovariectomized rats exerts lasting effects on cognitive function and the hippocampus.

We determined whether transient exposure to estradiol during middle age in ovariectomized rats would exert lasting effects on cognition and the brain beyond the period of exposure. Two experiments were conducted. Rats 10-11 months of age were ovariectomized and received vehicle control treatment throughout the experiment, continuous estradiol treatment throughout the experiment, or 40 d of transient exposure to estradiol that ended 3 d before behavioral training. In the first experiment, rats were trained on a radial-maze working memory task and killed 2 months after the termination of transient exposure to estradiol. The hippocampus was immunostained for choline acetyltransferase and estrogen receptors alpha (ER alpha) and beta (ER beta) by Western blotting. In a second experiment to determine the durability of treatment effects, rats were behaviorally tested every other month until brains were collected for Western blotting 8 months after the termination of transient exposure to estradiol. Maze testing included delay trials and scopolamine trials, in which dose-effect curves for the muscarinic receptor antagonist were determined. Transient exposure to estradiol enhanced working memory and attenuated amnestic effects of scopolamine as effectively as continuous estradiol exposure. Enhancements persisted for up to 7 months. Transient exposure to estradiol increased hippocampal levels of ER alpha and choline acetyltransferase 2 months and ER alpha 8 months after termination of the exposure. Neither estradiol treatment altered estrogen receptor beta levels. Results demonstrate that short-term treatment with estradiol during middle age enhances working memory well beyond the duration of treatment and suggest ER alpha as a potential mechanism for this effect.

The beneficial effects of estradiol on attentional processes are dependent on timing of treatment initiation following ovariectomy in middle-aged rats.

The goal of the present study was to explore the effects of long-term hormone deprivation on the ability of subsequent estrogen treatment to affect attention performance on the 5-choice serial reaction time task (5-CSRTT). In an initial experiment to assess estradiol effects in young adults, 2-month-old rats were trained on the 5-CSRTT, then ovariectomized and immediately implanted with capsules containing cholesterol (n=10) or estradiol (n=10). Then rats were tested on the 5-CSRTT under baseline task parameters, under increased task difficulty (behavior challenge condition), and finally in muscarinic and nicotinic drug challenge conditions. In a second experiment, 10-month-old rats were trained on the 5-CSRTT and at 12 or 17 months of age rats were ovariectomized and treated with estradiol or cholesterol, so that one group received continuous cholesterol control treatment, two groups received estradiol treatment immediately following ovariectomy (either at 12 or 17 months), and one group received delayed estradiol treatment initiated 5 months following ovariectomies. At 17 months of age, rats were tested on the 5-CSRTT. Baseline performance was comparable between estradiol- and cholesterol-treated rats of both age groups. However, young estradiol-treated rats outperformed controls when behavior was challenged by shortening the intertrial interval (Short ITI). In the same Short ITI condition, middle-aged rats receiving immediate estradiol treatment beginning at the age of 17 months, but not 12 months, outperformed controls as well as animals receiving delayed estradiol treatment. No differences between groups were found in the cholinergic drug challenge conditions. These data indicate that chronic estradiol treatment for approximately 1 month but not 6 months is able to enhance attention performance, and that prolonged ovarian hormone deprivation attenuates these beneficial effects of subsequent estradiol treatment. These findings have implications for informing clinical research about the importance of timing and duration of hormone treatment.

Increased daily handling of ovariectomized rats enhances performance on a radial-maze task and obscures effects of estradiol replacement.

Estrogen impacts performance on tasks of learning and memory, although there are inconsistencies in the direction and magnitude of the reported effects. Contributory factors to the inconsistencies may be methodological differences associated with different regimens of treatment. The goal of the present experiment was to assess the effect of increased handling, such as that commonly associated with pharmacological or other experimental manipulations, on the ability of estrogen to influence working memory performance. Young adult rats were ovariectomized and implanted with capsules containing either cholesterol or 25% estradiol diluted in cholesterol. Half of each hormone treatment group received standard handling, which consisted of handling required to carry out experimental procedures and half received increased handling, which consisted of standard handling as well as 2 min of additional daily handling by the experimenter. Animals were trained daily on a working memory task on an eight-arm radial maze for 24 days of acquisition and for eight additional daily trials in which delays of either 1 min or 3 h were imposed between the fourth and fifth arm choices. Animals that received increased handling exhibited significantly enhanced performance during acquisition and delay trials compared to those that received standard handling. Estradiol significantly enhanced performance during delay trials in animals that received standard handling but had no effect in animals that received increased handling. These results suggest that the amount of handling that animals receive as part of experimental procedures may obscure the memory enhancing effects of estradiol replacement on certain tasks of cognition.