Veronica M. Porterfield

Identification of novel light-induced genes in the suprachiasmatic nucleus

BackgroundThe transmission of information about the photic environment to the circadian clock involves a complex array of neurotransmitters, receptors, and second messenger systems. Exposure of an animal to light during the subjective night initiates rapid transcription of a number of immediate-early genes in the suprachiasmatic nucleus of the hypothalamus. Some of these genes have known roles in entraining the circadian clock, while others have unknown functions. Using laser capture microscopy, microarray analysis, and quantitative real-time PCR, we performed a comprehensive screen for changes in gene expression immediately following a 30 minute light pulse in suprachiasmatic nucleus of mice.ResultsThe results of the microarray screen successfully identified previously known light-induced genes as well as several novel genes that may be important in the circadian clock. Newly identified light-induced genes include early growth response 2, proviral integration site 3, growth-arrest and DNA-damage-inducible 45 beta, and TCDD-inducible poly(ADP-ribose) polymerase. Comparative analysis of promoter sequences revealed the presence of evolutionarily conserved CRE and associated TATA box elements in most of the light-induced genes, while other core clock genes generally lack this combination of promoter elements.ConclusionThe photic signalling cascade in the suprachiasmatic nucleus activates an array of immediate-early genes, most of which have unknown functions in the circadian clock. Detected evolutionary conservation of CRE and TATA box elements in promoters of light-induced genes suggest that the functional role of these elements has likely remained the same over evolutionary time across mammalian orders.

Repeated stressor exposure regionally enhances beta-adrenergic receptor-mediated brain IL-1β production.

It has been proposed that increased brain cytokines during repeated stressor exposure can contribute to neuropathological changes that lead to the onset of depression. Previous studies demonstrate that norepinephrine acting via beta-adrenergic receptors (β-ARs) mediate brain IL-1 production during acute stressor exposure. The aim of the current studies was to examine how the regulation of brain cytokines by adrenergic signaling might change following repeated stressor exposure. Fischer rats were exposed to four days of chronic mild stress and 24h after the last stressors β-AR expression, norepinephrine turnover, and β-AR-mediated induction of brain IL-1 were measured in limbic areas (e.g. hypothalamus, hippocampus, amygdala, and prefrontal cortex) and brainstem. Repeated stressor exposure resulted in decreases in β-AR expression (B(max)) measured by saturation binding curves in many limbic brain areas, while an increase was observed in the brainstem. This coincided with significant increases in norepinephrine turnover in the prefrontal cortex, hypothalamus, and amygdala, a significant increase in norepinephrine turnover was not observed in the hippocampus or brainstem. Stress increased overall IL-1 production in the amygdala (both basal and stimulated). While stress did not affect basal IL-1 levels in any other brain area, central administration of isoproterenol (a β-AR agonist) augmented IL-1 production in the hypothalamus of stressed animals. These data indicate that repeated stressor exposure results in brain area specific enhancements in β-AR-mediated IL-1 production and extends current knowledge of stress-induced enhancement of brain cytokine beyond sensitized response to immunological stimuli.

Time-dependent mediators of HPA axis activation following live Escherichia coli

The hypothalamus-pituitary-adrenal (HPA) axis is activated during an immune challenge to liberate energy and modulate immune responses via feedback and regulatory mechanisms. Inflammatory cytokines and prostaglandins are known contributors to HPA activation; however, most previous studies only looked at specific time points following LPS administration. Since whole bacteria have different immune stimulatory properties compared with LPS, the aim of the present studies was to determine whether different immune products contribute to HPA activation at different times following live Escherichia coli challenge. Sprague-Dawley rats were injected intraperitoneally with E. coli (2.5 × 10(7) CFU) and a time course of circulating corticosterone, ACTH, inflammatory cytokines, and PGE(2) was developed. Plasma corticosterone peaked 0.5 h after E. coli and steadily returned to baseline by 4 h. Plasma PGE(2) correlated with the early rise in plasma corticosterone, whereas inflammatory cytokines were not detected until 2 h. Pretreatment with indomethacin, a nonselective cyclooxygenase inhibitor, completely blocked the early rise in plasma corticosterone, but not at 2 h, whereas pretreatment with IL-6 antibodies had no effect on the early rise in corticosterone but attenuated corticosterone at 2 h. Interestingly, indomethacin pretreatment did not completely block the early rise in corticosterone following a higher concentration of E. coli (2.5 × 10(8) CFU). Further studies revealed that only animals receiving indomethacin prior to E. coli displayed elevated plasma and liver cytokines at early time points (0.5 and 1 h), suggesting prostaglandins suppress early inflammatory cytokine production. Overall, these data indicate prostaglandins largely mediate the early rise in plasma corticosterone, while inflammatory cytokines contribute to maintaining levels of corticosterone at later time points.

Temporal patterns of light-induced immediate-early gene expression in the suprachiasmatic nucleus

Exposing an animal to light during the normal dark period of its daily cycle induces shifts in the animals circadian rhythm of activity. These shifts are preceded by an increase in the expression of an array of immediate early genes in the suprachiasmatic nucleus, the location of the primary circadian clock in the brain. For most of these genes, little is known about the physiological significance of their expression in the SCN. In order to characterize the expression of these genes, laser capture microscopy, and real-time PCR were used to measure the time course of expression of immediate-early genes in the SCN after a 30-min light pulse during the early portion of the night. Most of the measured genes show peak expression shortly after the end of the stimulus and then decline back to baseline after 2h. However, a few genes, including Rrad, Egr3, and Jun, show a more sustained elevation in expression. Analysis of the function of light-induced genes in other cellular systems suggests a possible role for these genes in reducing the SCN to subsequent photic stimuli and in protecting the SCN from excitotoxicity.

Changes in the noradrenergic system following chronic stress: Implications for brain cytokine production

In the clinical setting, minimizing the extent of oncological surgeries is believed to reduce cancer recurrence. We reported that blocking excess release of catecholamines and prostaglandins in the perioperative context attenuates suppression of anti-cancer immunity and prevents promotion of experimental and spontaneous metastasis by surgery. Here we study colon cancer, comparing several approaches of inducing experimental liver metastasis, employing different surgical and inoculation procedures, and attempting to minimize surgical stress. Syngeneic CT26 colorectal cancer cells were administered to BALB/c female and male mice, either through the hepatic portal vein, or through different approaches of intrasplenic inoculation. Surgical severity of the inoculation procedure was manipulated. Vehicle treated mice were compared to those treated with the beta-blocker, propranolol, and the COX-2 inhibitor, etodolac. Results: Males were significantly more susceptible than females to tumor development (metastases number and liver weight). Retaining the entire spleen (portal inoculation) or removing hemispleen or the entire spleen (intra-splenic inoculation), did not affect resistance to metastasis. Increasing the surgical severity, by adding laparotomy or by extending surgical time and hypothermia, did not worsen metastatic outcomes. Combined administration of propranolol + etodolac significantly improved metastatic resistance irrespective of surgical severity and inoculation procedure. Thus, employing propranolol + etodolac along the perioperative period could be advantageous in both mild and severe surgical procedures in colorectal cancer, especially given the expected pre-surgical psychological stress responses and tumor release of prostaglandins.

Time-Dependent Mediators of HPA Axis Activation Following Live E.coli

The hypothalamus-pituitary-adrenal (HPA) axis is activated during an immune challenge to liberate energy and modulate immune responses via feedback and regulatory mechanisms. Inflammatory cytokines and prostaglandins are known contributors to HPA activation; however, most previous studies only looked at specific time points following LPS administration. Since whole bacteria have different immune stimulatory properties compared with LPS, the aim of the present studies was to determine whether different immune products contribute to HPA activation at different times following live Escherichia coli challenge. Sprague-Dawley rats were injected intraperitoneally with E. coli (2.5 × 10(7) CFU) and a time course of circulating corticosterone, ACTH, inflammatory cytokines, and PGE(2) was developed. Plasma corticosterone peaked 0.5 h after E. coli and steadily returned to baseline by 4 h. Plasma PGE(2) correlated with the early rise in plasma corticosterone, whereas inflammatory cytokines were not detected until 2 h. Pretreatment with indomethacin, a nonselective cyclooxygenase inhibitor, completely blocked the early rise in plasma corticosterone, but not at 2 h, whereas pretreatment with IL-6 antibodies had no effect on the early rise in corticosterone but attenuated corticosterone at 2 h. Interestingly, indomethacin pretreatment did not completely block the early rise in corticosterone following a higher concentration of E. coli (2.5 × 10(8) CFU). Further studies revealed that only animals receiving indomethacin prior to E. coli displayed elevated plasma and liver cytokines at early time points (0.5 and 1 h), suggesting prostaglandins suppress early inflammatory cytokine production. Overall, these data indicate prostaglandins largely mediate the early rise in plasma corticosterone, while inflammatory cytokines contribute to maintaining levels of corticosterone at later time points.