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.

Prenatal stress influences 8-OH-DPAT modulated startle responding and [3H]-8-OH-DPAT binding in rats.

The present study was to investigate some aspects of the 5-HT1A receptor system in adult-aged rats (50-60 days) that were either exposed to prenatal stress (PS) or not exposed to prenatal stress (CON). In the first series of experiments, rats were pretreated with vehicle, the 5-HT1A agonist 8-OH-DPAT or the 5-HT1A antagonist, WAY-100635 and exposed to 120 acoustic startle stimuli (95 dB) using a 30 s inter-trial interval. 8-OH-DPAT produced a dose-dependent increase in acoustic startle responding in CON and PS rats, with the PS rats exhibiting greater responding than CON rats. WAY-100635 depressed startle amplitudes only in the CON group. Finally, radioligand binding studies using [3H]-8-OH-DPAT indicated a significant decrease in receptor density in hippocampal homogenates from PS rats but no difference in [3H]-8-OH-DPAT binding from homogenates of the amygdala. Our results are consistent with earlier reports indicating that prenatal stress alters the serotonergic system. Specifically, our results indicate that gestational exposure to chronic mild stress enhances startle amplitudes following 8-OH-DPAT administration, prevents the depression in startle amplitudes following WAY-100635 administration and reduces [3H]-8-OH-DPAT binding in hippocampal preparations.

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.

The differential effects of prenatal stress in rats on the acoustic startle reflex under baseline conditions and in response to anxiogenic drugs.

RATIONALE: The prenatal stress syndrome (PS) is characterized by exaggerated behavioral and physiological responses to stressful stimuli and anxiogenic agents. OBJECTIVES: To characterize the behavioral effects of PS on the acoustic startle reflex (ASR) and to determine the possible role of PS-induced alterations in noradrenergic control of ASR by determining the effects of the alpha2-adrenoceptor antagonists, yohimbine, idazoxan, and RS79948-197. METHODS: PS was induced by exposing pregnant dams to a mild stressor of handling and saline injection (0.1 ml, s.c.) from gestational days 14 to 21. Control dams were left undisturbed throughout pregnancy. Using adult male offspring, all ASR studies consisted of either a 30- or 60-min testing period containing 60 or 120 acoustic startle stimuli trials (95 dB, 50 ms noise burst) at a fixed intertrial interval of 30 s after a 5-min acclimation period. For drug studies, a 3-day repeated measures design was implemented. RESULTS: With the exception of the response to the first startle stimulus on the first day of testing, there were no significant differences in baseline ASR between control and PS offspring. Low doses of yohimbine, idazoxan, and RS79948-197 were anxiogenic in the ASR test in both control and PS offspring. PS offspring were less responsive to higher doses of yohimbine (5 mg/kg) and idazoxan (8 mg/kg) but did not differ from control in their responses to any dose of RS79948-197. CONCLUSIONS: Anxiogenic effects of yohimbine, idazoxan, and RS79978-197, likely mediated via alpha2-adrenoceptor blockade, are similar in control and PS rats. Differences between control and PS rats occurred in the response to higher doses of yohimbine and idazoxan. Non-specific effects of these drugs, such as actions at 5HT1A receptors, may cause their behavioral profile to be altered by PS, and to differ from the highly selective RS79948-197.

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.

N-methyl-D-aspartate (NMDA)-mediated corticotropin-releasing factor (CRF) release in cultured rat amygdala neurons.

Corticotropin-releasing factor (CRF) plays an important role in the activation of centrally mediated responses to stress. The amygdala, a limbic structure involved in the stress response, has a significant number of CRF cell bodies and CRF receptors. Activation of glutamatergic projections to the amygdala has been implicated in the stress response. Few studies have evaluated neurotransmitter-stimulated CRF release in the amygdala. We measured the effects of glutamate (0.1-1000 microM) and N-methyl-D-aspartate (NMDA, 0.1-1000 microM) on CRF release from the amygdala using primary neuronal cultures from embryonic rat brains (E18-19). Experiments were performed after the cultures grew for 17-20 days. CRF was measured using radioimmunoassay. The excitatory amino acid neurotransmitters, glutamate and NMDA, stimulated CRF release in a concentration-dependent manner. The apparent EC50 values for glutamate and NMDA were 17.5 microM and 12 microM, respectively. Consistent with a NMDA receptor-driven event, glutamate-stimulated CRF release was blocked by the NMDA antagonist, 2-amino-5-phosphonovaleric acid (AP-5, 1-100 microM) and antagonized by the addition of 1.2 mM MgCl2 to the incubation medium. These results implicate an inhibition of CRF release in the amygdala as a possible mechanism for the reported anxiolytic effects of NMDA antagonists.

Corticotropin-releasing factor and defensive withdrawal: inhibition of monoamine oxidase prevents habituation to chronic stress.

There is growing evidence for a role of extrahypothalamic corticotropin-releasing factor (CRF) in the pathogenesis of anxiety. A modified form of the defensive withdrawal test was used to test the anxiogenic effects of acute administration of intracerebroventricular (1 microg, i.c.v.) CRF in adult male rats. Habituation to the mild stress of daily handling and subcutaneous (s.c.) saline injection over 2-6 weeks abolished the anxiogenic effects of exogenous CRF. At 6 weeks this habituation also resulted in attenuation of baseline withdrawal behavior. CRF receptor binding was significantly decreased in the amygdala of chronically handled animals and may have been responsible for this habituation phenomenon. Comparison of rats treated with the monoamine oxidase (MAO) inhibitor, phenelzine [3 mg/kg, s.c., daily for 2-6 weeks] to the saline-treated groups revealed a failure to habituate to the chronic handling, as the baseline withdrawal (after injection of artificial CSF) by the phenelzine-treated animals was not different from the baseline withdrawal by unhandled rats. In comparison to rats treated chronically with saline, phenelzine treatment enhanced the anxiogenic effect of CRF. In summary, habituation to a mild chronic stress decreased baseline defensive withdrawal. Intraventricular administration of CRF produced an anxiogenic response as measured in the defensive withdrawal test, which was lost through exposure to mild chronic stress. Two or 6 weeks of daily handling and SC saline injection caused a downregulation of CRF receptors in the amygdala, which could account for the behavioral habituation and the loss of CRF-induced defensive withdrawal. Phenelzine treatment concurrent with mild chronic stress prevented habituation and maintained the anxiogenic effect of CRF in spite of the downregulation of CRF receptors in the amygdala.

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.

Release of platelet activating factor (PAF) and eicosanoids in UVC-irradiated corneal stromal cells.

Ultraviolet (UV) irradiation provokes acute inflammation of the eye, and can be used to model processes that occur in response to damage to the anterior segment. This study characterized ultraviolet-C (UVC, 254 nm) irradiation-induced PAF synthesis, and arachidonic acid (20:4) and eicosanoid release in rabbit corneal stromal cells maintained in vitro. PAF was measured by radioimmunoassay (RIA) after exposing cultured corneal stromal cells to UVC irradiation (20 min, 2, 5, 10 mW/cm2). 14C-20:4-labeled stromal cells were also stimulated with UVC and radiolabeled phospholipids, neutral lipids and eicosanoids were measured. Synthesis of cell-associated and secreted PAF from corneal stromal cells was increased by UV irradiation. UV irradiation (254 nm, 5mW/cm2) enhanced 20:4 release from triacylglycerols, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine, and increased levels of 20:4-diacylglycerol and unesterified 20:4. The released 20:4 entered both the cyclooxygenase and lipoxygenase pathways after UVC irradiation. The PAF antagonist, BN52021 (10 microM) reduced UVC irradiation-induced stimulation of prostaglandin production, but failed to inhibit UVC-induced 20:4 release and synthesis of lipoxygenase products. Furthermore, exogenous PAF (1 microM) stimulated prostaglandin production, but did not increase the synthesis of lipoxygenase products from radiolabeled 20:4. The effects of PAF on prostaglandin synthesis were inhibited by BN52021. These findings indicate that responses to injury in cultured corneal stromal cells include PAF synthesis, release of 20:4 from glycerolipids, accumulation of diacylglycerol and synthesis of eicosanoids. The data further suggest that during UVC irradiation in vitro, PAF is not a primary or initial mediator of 20:4 release and synthesis of lipoxygenase products, but may mediate UVC-induced prostaglandin synthesis.

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells.

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 17 beta-estradiol, or dexamethasone on [1-14C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-14C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-14C]AA was diminished by 96-h treatment with 1,25(OH)2D3 (79 +/- 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 +/- 2% of control, P < 0.05). The 1,25(OH)2D3-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-14C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)2D3 was without effect on either total [1-14C]AA uptake or the specific [1-14C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)2D3 treatment decreased hOB cell uptake of [1-14C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)2D3-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17 beta-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.

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.

In vivo microdialysis of corticotropin releasing factor (CRF): calcium dependence of depolarization-induced neurosecretion of CRF.

Corticotropin releasing factor (CRF) is a large neuropeptide which functions as a major neurotransmitter in physiological stress responses. We have developed a microdialysis method for detecting CRF release from the median eminence of anesthetized rats. Depolarizing concentrations of KCl increased release of CRF into the perfusion media; this effect was inhibited by 100 microM verapamil. Our characterization of the physiologic conditions of KCl-induced release of neuronal CRF using the microdialysis technique provides evidence that the CRF release system is Ca(2+)-dependent and maintains its integrity over many hours in anesthetized rats.

Dietary fish oil administration retards blood pressure development and influences vascular properties in the spontaneously hypertensive rat (SHR) but not in the stroke prone-spontaneously hypertensive rat (SHR-SP).

In the present study, we compared the blood pressure in the SHR-SP and in the spontaneously hypertensive rat (SHR) after dietary administration of fish oil from 4 to 17 weeks of age. The retarding influence of dietary fish oils on the development of hypertension was prominent in the SHR (26 mmHg) and not evident in the SHR-SP (8 mmHg). The enhanced development of blood pressure in both the SHR and the SHR-SP is characterised by an elevated maximum contraction in the mesenteric vascular bed to sympathetic nerve stimulation and to injected noradrenaline. In SHR, but not SHR-SP, this maximum contraction was significantly attenuated by dietary fish oil. Likewise, acetylcholine mediated relaxation of the isolated aorta was enhanced in preparations from the SHR but not the SHR-SP. These physiological changes were also associated with a change in the total n-3 polyunsaturated fatty acids (PUFAs) content in vascular tissue, which were inversely proportional to the prevailing blood pressure values seen in all three strains of rat receiving dietary fish oils. Platelet activated thromboxane production was equally depressed in WKY (Wistar Kyoto), SHR and SHR-SP rats. The results indicate that the blood pressure lowering effect of fish oil when administered during the period of development of hypertension is much greater in the SHR than it is in the SHR-SP. Furthermore the lowering of blood pressure by fish oil administration is related to a restoration of normal vascular contraction and normal vascular relaxation, but not related to a suppression of serum thromboxane production.

Depolarization-induced release of corticotropin-releasing factor (CRF) in primary neuronal cultures of the amygdala.

The 41-amino acid neuropeptide, corticotropin-releasing factor (CRF) is distributed throughout the central nervous system and appears to play a pivotal role in stress, anxiety and depression. CRF is present in high concentrations in the limbic brain region, the amygdala, an area important in emotional and autonomic responses to stress. In this report, primary neuronal cultures of amygdala from fetal rat brains (E18-E19) were used to study depolarization-induced CRF release. Immunocytochemical analyses of the cultures revealed a bead-like distribution of CRF immunoreactivity (CRFir) in about 1% of the neurons. Time course studies showed that 56 mM KCl-evoked CRF release occurred with an initial burst during the first minute that was maintained over 30 min; basal CRF release slightly increased over a 30-min period. CRF release in response to depolarization increased with increasing cell density and with increasing days in culture. Multiple serial incubations alternating basal and depolarizing conditions caused a depletion of the releasable pool of CRF. Potassium-evoked CRF release was calcium-dependent. These data suggest that primary neuronal cultures of fetal rat amygdala are an effective model system to study CRF release in this brain region.

Regional and temporal variations in the accumulation of unesterified fatty acids and diacylglycerols in the rat brain during kainic acid induced limbic seizures.

These experiments tested the hypothesis that limbic seizures induced by kainic acid (KA) activate mechanisms (e.g. phospholipase) that degrade the cell membrane, causing a release and accumulation of free fatty acids (FFAs) and diacylglycerols (DGs) in brain areas susceptible to seizure-related damage. The possible link between these effects on lipids and the subsequent development of seizure-related brain damage was investigated by studying the temporal and regional relationship between alterations in lipids in the hippocampus, frontal cerebral cortex, amygdala, striatum and cerebellum, and the development and severity of seizures. Rats were treated with 10 mg/kg KA (s.c.) and sacrificed by head focused microwave irradiation at 1 h, 2 h, 24 h, or 7 days. Levels of FFAs and DGs were determined by gas liquid chromatography (GLC). Brain regions from control rats differed markedly in the content and composition of both FFA and DG pools. Changes in FFAs and DGs during KA-induced limbic seizures also varied from region to region and over time after drug treatment. The largest increases in FFAs in amygdala, striatum, cortex and hippocampus occurred during the peak of seizure activity. Although DG levels were altered in some areas at some time points, there was no apparent correlation between changes in DGs and seizure severity. However, increases in DGs occurred at later time points, coincident with the occurrence of neuronal cell loss in amygdala, cortex, hippocampus and striatum. These data indicate that limbic seizures activate the accumulation of FFAs through increased neuronal activity, while accumulation of DGs may be related to the development of seizure-related brain damage.

Abnormal fatty acid composition in sarcolemma and sarcoplasmic reticulum from myotonic ADR mouse muscle.

The fatty acid composition of membrane lipids from sarcolemma and sarcoplasmic reticulum isolated from biceps and gastrocnemius muscle has been compared in normal (wildtype, +/adrmto or +/+) and affected (adrmto/adrmto) myotonic mice. The adrmto mouse exhibits an arrested development of the righting response, and arose spontaneously from the SWR/J strain. These mice exhibit classical myotonia similar to the human disease, Becker's myotonia [1]. Significant alterations, characterized by a decrease in the saturated fatty acid, palmitic acid (16:0), and the polyunsaturated fatty acid, arachidonic acid (20:4), and an increase in stearic (18:0) and linoleic (18:2) acids, were observed between sarcolemma and sarcoplasmic reticulum from normal and affected mice. These changes in fatty acid composition of muscle membrane from ADR mice may be adequate to cause an alteration in membrane fluidity and affect the function of ion channels. The fatty acid composition of erythrocytes ghosts was also examined, as a potential marker for alterations in muscle membranes. In erythrocyte ghosts isolated from affected mice, the only alteration observed was a decrease in the proportion of oleic acid (18:1), an effect completely different from those observed in muscle membranes. Therefore, erythrocyte ghosts do not serve as an adequate indicator of changes in fatty acid composition of muscle membranes in this model of myotonia.

Intracamerally injected platelet activating factor (PAF) induces marked intraocular inflammatory reactions.

An inflammatory response was elicited in the rabbit eye by intracameral injection of platelet activating factor (PAF). PAF induced severe aqueous flare, corneal edema, pupillary constriction and marked biphasic changes in intraocular pressure (IOP) in a dose-dependent manner. All of the responses to PAF were inhibited by the PAF receptor antagonist, BN 52021 (20 mg/kg, i.p.). The cyclooxygenase inhibitor, indomethacin (30 mg/kg, i.p.) caused significant inhibition of the early phase PAF-induced aqueous flare, pupillary constriction and intraocular hypertension, but did not effect PAF-induced corneal edema or intraocular hypotension. NDGA (10 mg/kg, i.p.), a lipoxygenase inhibitor, did not inhibit the inflammatory effects of PAF. PAF-induced chemotactic response was evaluated by tissue chemiluminescence. Intracamerally injected PAF did not significantly increase chemiluminescence in cornea or iris-ciliary body, but intracorneal injection of PAF did cause a chemotactic response in both the conjunctiva and cornea. These data suggest that PAF may be an important mediator of intraocular inflammation and that some PAF-induced effects are prostaglandin dependent, while others may be independent of eicosanoid synthesis and release.

Reciprocal regulation of fatty acid release in the brain by GABA and glutamate.

Several model systems have been used to test the hypothesis that the release of FFA in the brain is regulated by depolarization of neurons. This FFA release is likely the result of the activation of phospholipase A2. The increased neuronal activity that occurs due to synchronous depolarization during seizures causes activation of phospholipase A2. Decreasing neuronal activity by administering the anxiolytic, diazepam, appears to decrease the activity of phospholipase A2. The GABA antagonist, bicuculline, which causes depolarization by negating the hyperpolarizing tone imposed on neurons by GABA, causes FFA release in synaptosomes and in neurons in tissue culture. Likewise, the glutamate agonist, kainic acid, which depolarizes neurons by opening sodium channels, increases the activity of phospholipase A2. PC-specific phospholipase C, another enzyme important in the generation of the second messenger, DG, is also activated by depolarization. Several important questions remain to be answered. The site of FFA release, in terms of the pre-vs. postsynaptic membrane, is not clear, although the experiments with synaptosomes support the hypothesis that activation of phospholipase A2 may be an important regulator of presynaptic events. This idea has also been suggested by studies on the phenomenon of long-term potentiation, where free 20:4 or its metabolites may be involved in presynaptic facilitation of neurotransmitter release (Freeman et al., 1990; Massicotte et al., 1990; Williams et al., 1989; also see Dorman, this volume). The activation of the PI cycle and subsequent stimulation of protein kinase C may be a postsynaptic event important in the integration of inputs at the dendrite and soma or a presynaptic event involved in the modulation of neurotransmitter release (Taniyama et al., 1990; El-Fakahany et al., 1990; also see Nishizuka, this volume). Therefore the stimulation of a PC-specific phospholipase C, which is capable of generating large amounts of DG over a prolonged period of time (Exton, 1990; Martinson et al., 1990; Diaz-Laviada et al., 1990), could occur at either site. Another important question is the role of FFA and DG in affecting cell-cell signaling events, particularly with regard to ion fluxes. Modulation of an acetylcholine-linked K+ channel in the heart by FFA and their oxygenation products has been reported (Kim and Clapham, 1989). The cardiac muscarinic receptor is linked to a hyperpolarizing K+ channel via a G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Bicuculline induces free fatty acid release from phospholipids in neuro-2A cells in culture.

This study characterizes free fatty acid release in a neuroblastoma cell line (Neuro-2A), a potential model system for the study of factors that control phospholipase A2 in neurons. Two compounds, bicuculline (an antagonist at gamma-aminobutyric acid receptors), and A23187 (a Ca2+ ionophore), were examined. The release of endogenous fatty acids and the turnover of radiolabeled arachidonic and docosahexaenoic acids were measured. The cells actively incorporated radiolabeled fatty acids into various glycerolipid pools. Both endogenous fatty acids and radiolabeled fatty acids were released from glycerolipids in a time-dependent manner. Phosphatidylcholine was a major source of released fatty acids. Release of free fatty acids was markedly stimulated by both bicuculline and A23187. We conclude that the Neuro-2A cells contain phospholipase activity that is sensitive to Ca2+ ionophore and bicuculline, and may provide a good system for further studies on the regulation of phospholipase A2 in neurons.

Increased levels of leukotriene C4 in retinal pigment epithelium are correlated with early events in photoreceptor shedding in Xenopus laevis.

The levels of 5-lipoxygenase products of arachidonic acid, leukotriene (LT) B4 and LTC4 in retinal pigment epithelia (RPE) from Xenopus laevis were measured by radioimmunoassay (RIA). RPE were isolated during various stages of photoreceptor renewal to determine possible alterations in 5-lipoxygenase activity concurrent with photoreceptor detachment and phagocytosis. Both LTC4 and LTB4 were released to RPE incubation media, although levels of LTB4 in unstimulated RPE were close to the limits of detection by RIA. Incubation of RPE with the calcium ionophore A23187 increased the levels of both LTB4 and LTC4. When animals were maintained on a cycle of 12 hr light/dark, normal photoreceptor shedding, as measured by histological quantitation of the appearance of phagosomes in the RPE, occurred 1 hr after light onset. Levels of LTC4 in RPE were lower 1 hr after light onset, as compared to 1 hr prior to light onset. Due to the low levels of LTB4, no significant differences could be detected. However, when LTB4 levels were elevated with A23187, LTB4 also declined 1 hr after light onset. When animals were maintained in constant light for 5 days, then exposed to 2 hr dark and 2 hr light, a massive shedding response occurred. Levels of LTC4 were stimulated 5 min after light onset (prior to detectable shedding) and declined below dark levels as shedding progressed. These data suggest a correlation between 5-lipoxygenase activity and the events of photoreceptor shedding and phagocytosis.