Dale L. Birkle

Prenatal stress increases corticotropin-releasing factor (CRF) content and release in rat amygdala minces

Corticotropin-releasing factor (CRF) is a neuropeptide found throughout the central nervous system that has a proposed role in modulating emotional and behavioral states, including stress and anxiety. The amygdala, which is important in the control of emotional and autonomic responses to stress, contains CRF nerve terminals, CRF cell bodies, and CRF receptors. In rats, exposure to prenatal stress results in offspring that display a hyperemotional state and increased anxiety. In this study the effects of prenatal stress on CRF release was measured in amygdala minces (1 mm3) obtained from adult (8-16 weeks of age) male offspring of dams subjected to daily saline injection (0.1 ml, s.c.) from gestational day 14 to 21. CRF release from amygdala was time- and calcium-dependent, and stimulated by KCl-induced depolarization. Depolarization-induced CRF release was significantly increased by 42% from the amygdala of prenatally stressed offspring versus controls. Prenatally stressed offspring also showed a 49% increase in CRF levels in the amygdala. The increased amounts of CRF released in response to depolarization were likely the consequence of increased tissue content of CRF, as fractional release under basal or KCl-stimulated conditions was not different in the prenatal stress group versus control. This suggests that a long-lasting up-regulation of the CRFergic neurotransmission may occur in the amygdala, which may be important in the generation of hyperemotional offspring after exposure to prenatal stress.

Effects of prenatal stress on defensive withdrawal behavior and corticotropin releasing factor systems in rat brain

Exposure of pregnant rats to stress results in offspring that exhibit abnormally fearful behavior and have elevated neuroendocrine responses to novelty and aversive stimuli. This study examined the effects of prenatal stress on plasma corticosterone, adrenal weight, defensive withdrawal behavior, and the density of receptors for corticotropin releasing factor (CRF) in the amygdala. Pregnant Sprague-Dawley rats were stressed by daily handling and saline injection (s.c., 0.9%, 0.1 mL) during the last week of gestation. Male offspring were studied at adulthood (60-120 days of age). Adrenal hypertrophy and increased plasma corticosterone were observed in the prenatally stressed offspring. Defensive withdrawal, an ethological measure of the conflict between exploratory behavior and retreat, was quantified in naive offspring, and in offspring exposed to restraint stress (2 h). Restraint stress increased defensive withdrawal in both control and prenatally stressed offspring. Both naive and restraint-stressed prenatally stressed offspring exhibited increased defensive withdrawal compared to control offspring. There was a significant interaction between prenatal stress and restraint stress, suggesting increased vulnerability of prenatally stressed offspring. The effects of restraint in the defensive withdrawal test were reduced by intracerebroventricular administration of the CRF antagonists, alpha-helical CRF9-41 (20 microg every hour) or D-phe(12), Nle(21, 38), C(alpha)-MeLeu(37)]-CRF((12-41)) (5 microg every hour) during the restraint period. The difference between control and prenatally stressed offspring was abolished by the CRF antagonists, suggesting that increased activation of CRF receptors may be a factor in the behavioral abnormalities of prenatally stressed rats. Measurement of CRF receptors in amygdala revealed a 2.5-fold increase in binding in prenatally stressed offspring. In light of previous work from this laboratory demonstrating increased content and release of CRF in amygdala from prenatally stressed offspring, the present study suggests that the increased fearfulness of prenatally stressed rats may be a consequence of increased activity of CRFergic systems in the amygdala.

Docosahexaenoic acid (22:6, n-3) is metabolized to lipoxygenase reaction products in the retina

Docosahexaenoic acid (22:6, n-3), a major component of retinal phospholipids, is a substrate for active lipoxygenation in intact canine retinas incubated in vitro with [U-14C]docosahexaenoic acid. The major lipoxygenase reaction product was identified by high performance liquid chromatography and gas chromatography-mass spectrometry as 11-hydroxy-4,7,9-(trans)13,16,19 docosahexaenoic acid. Other mono- and di-hydroxy derivatives also were detected. The synthesis of these compounds was inhibited by the antioxidant and lipoxygenase inhibitor, nordihydroguaiaretic acid, but was not inhibited by indomethacin or esculetin.

Inflammation-induced stimulation of the synthesis of prostaglandins and lipoxygenase-reaction products in rabbit cornea

The cyclooxygenase and lipoxygenase pathways that produce prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) were studied in inflamed rabbit cornea. A cryogenic lesion was induced and five days later the epithelium, stroma and endothelium were isolated and incubated with [1-14C]arachidonic acid. After lesioning, the arachidonic acid metabolites, thromboxane B2, PGF2 alpha and 6-keto-PGF1 alpha exhibited the greatest increase in the stroma. Lipoxygenase products were formed in the three layers also, although 12-HETE predominated. The correlation between the synthesis of these compounds and corneal injury is discussed.

Effect of K+ depolarization on the synthesis of prostaglandins and hydroxyeicosatetra(5,8,11,14)enoic acids (HETE) in the rat retina. Evidence for esterification of 12-HETE in lipids.

[14C]Arachidonic acid is metabolized to prostaglandins and hydroxyeicosatetraenoic acids in the rat retina. After intravitreal injection of [14C]arachidonic acid, 25% of the injected radiolabel was recovered in the retinal lipids. Phosphatidylcholine and phosphatidylinositol were most actively labeled; however, all glycerolipids incorporated arachidonic acid. The synthesis of prostaglandins E2, F2 alpha, D2, 6-keto-F1 alpha, thromboxane B2 and hydroxyeicosatetraenoic acids was measured by high-performance liquid chromatography. The identity of 12-HETE was confirmed by gas chromatography-mass spectrometry. Incubation of prelabeled retinas in vitro promoted the release of [14C]arachidonic acid from glycerolipids. A 12-fold increase in the synthesis of hydroxyeicosatetraenoic acids occurred with no change in the synthesis of prostaglandins. Incubation in a depolarizing medium (45 mM K+) resulted in a selective increase in hydroxyeicosatetraenoic acids, an effect that was blocked by nordihydroguaiaretic acid (1 microM) and eicosatetraynoic acid (10 microM). 12-[3H8]Hydroxyeicosatetraenoic acid, intravitreally injected, was incorporated into retinal lipids with a distribution similar to arachidonic acid. When retinas labeled with 12-[3H8]hydroxyeicosatetraenoic acid were incubated, there was a large release of the incorporated radioactivity, and metabolism to other products with the chromatographic properties of dihydroxyeicosatetraenoic acids. The release of 12-hydroxyeicosatetraenoic acid was not affected by depolarizing conditions (45 mM K+); however, the conversion of 12-hydroxyeicosatetraenoic acid to dihydroxy isomers was stimulated by K+. These experiments demonstrate active pathways for the generation of eicosanoids in the rat retina that are sensitive to membrane depolarization and lipoxygenase inhibitors.

Mild prenatal stress in rats is associated with enhanced conditioned fear

We tested the hypothesis that prenatal stress would enhance conditioned fear in adult rats. Pregnant Sprague-Dawley rats were stressed by exposure to a novel environment and subcutaneous injection of saline (0.1 ml 0.9% NaCl) at random times daily from Days 14 to 21 of pregnancy. When compared to adult control (CON) male rats from unmanipulated pregnancies, adult prenatally stressed (PS) male rats showed increased freezing behavior in response to acute footshock as well as increased freezing behavior the next day in the same context, without shock delivery. In another experiment, the gestational stressor was examined for elevations in corticosterone and ACTH. At gestational days (G)15, G17, G19 and G21, maternal and fetal plasma was collected. Analysis showed elevations in corticosterone and ACTH in the PS dams when compared to the CON dams. Additionally, increased corticosterone was found in the PS fetuses when compared to the CON fetuses. Finally, some CON and PS litters were examined for alterations in length of gestation, number of pups born, bodyweight on postnatal day (P)1 and anogenital distance on P1 and differences were not found. In conclusion, our data demonstrate that a mild stressor during gestation, sufficient to raise plasma corticosterone and ACTH, is associated with enhanced conditioned fear during adulthood.

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina in vitro has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha and TXB2, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC50 = 8.3 nM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 microM), indomethacin (1 microM) and NDGA (IC50 = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.

Light exposure stimulates arachidonic acid metabolism in intact rat retina and isolated rod outer segments

This paper presents evidence that short-term exposure to light increases synthesis of hydroxyei-cosatetraenoic acid (HETE) and prostaglandin D2 (PGD2) and stimulates the uptake and metabolism of 20∶4 in phospholipids and triacylglycerols in rat retina. There was a time-dependent increase in incorporation of 1-14C-20∶4 into glycerolipids in both dark-adapted and light-exposed groups. Exposure to light for 15 or 30 min enhanced the acylation of 20∶4 into phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and triacylglycerols. In the light-exposed groups there was a large increase in the conversion of 20∶4 to leukotriene B4, two diHETEs, 5-HETE, 15-HETE, and PGD2. The stimulation of HETE synthesis by light could be due to early stages of light-induced lipid peroxidation in visual cells. To examine this, we studied peroxidation of 20∶4 in isolated rod outer segments (ROS). There was more oxidation of 20∶4 in light-exposed ROS, as compared to ROS incubated in the dark. Vitamin E and nordihydroguaiaretic acid inhibited the light-induced formation of some of these products. The data indicate that photo-oxidation of 20∶4 in ROS is accompanied by enzymatic oxygenation that is stimulated by light. Increased production of HETEs and PGD2 may be a consequence of the light-induced stimulation of the metabolism of 20∶4 in membrane phospholipids in the retina.

Early emergence of increased fearful behavior in prenatally stressed rats

We have been studying the mildly prenatally stressed (PS) rat as a potentially useful animal model of anxiety disorders. Previously we have demonstrated that there are anatomical and biochemical alterations in the amygdalas of adult PS offspring and that these offspring show increased fearful behaviors. However, human data indicate that anxiety disorders often present first in early childhood and then persist throughout adolescence and adulthood. To determine if PS rats also model this characteristic of human anxiety disorders, here we asked whether behavioral indices of increased fear would be detectable at an early age. We tested the hypotheses that young PS rats would show increased behavioral fearfulness in response to an acute stressor and that this would increase with age. A mild prenatal stressor, consisting of removal of the dam from the home cage and administration of a subcutaneous injection of 0.1 ml of 0.9% saline daily, was administered during the last week of pregnancy. Offspring were tested in the defensive-withdrawal apparatus before and after exposure to restraint stress at 25, 45 and 60 days of age. PS animals showed increased defensive-withdrawal behavior following the stressor and were more fearful following restraint when compared to controls (CON). This was significant at P45 and increased to P60. Hence, fearful behaviors in PS rats emerge prior to sexual maturation and increase in magnitude thereafter, further validating our model as a means to investigate the underpinnings of anxiety disorders.

Stimulation of the Amygdala by Glutamate Facilitates Corticotropin-Releasing Factor Release from the Median Eminence and Activation of the Hypothalamic-Pituitary-Adrenal Axis in Stressed Rats

The role of the amygdala in the regulation of hypothalamic release of corticotropin-releasing factor (CRF) was investigated. Microinjection of glutamate (50 nmol) into the amygdala resulted in increased plasma corticosterone in male rats previously subjected to a 14-day unpredictable stressor paradigm (p < or = 0.05 vs. saline-injected controls). A long-lived increase in corticosterone levels was also observed in rats which were urethane-anesthetized (1.35 g/kg) 3 h prior to glutamate microinjection (p < or = 0.01 vs. saline-injected controls). These effects on plasma corticosterone were observed despite the presence of high basal levels of corticosterone. Furthermore, microperfusion of glutamate (3-300 microM) into the amygdala of urethane-anesthetized rats resulted in a dose-dependent increase in CRF release from the median eminence, as assessed by in vivo microdialysis (p < or = 0.025 vs. basal). These results indicate a facilitating role for the amygdala in stress-induced increases in CRF release and subsequent adrenocortical activation.