A summary of ethanol effects at MOP and plasma membrane lipid dynamics compared to the effects of specific ligands.

*<p>Naltrexone and naloxone promote MOP association into larger molecular complexes.</p>**<p>Ethanol dissipates the plasma membrane micro-domains, thereby releasing the “associated” form and rendering “free” MOP.</p

Schematic presentation of the instrumentation for FCS/CLSM.

<p>To induce fluorescence, the sample is illuminated by incident laser light. The irradiating laser beam is reflected by a dichroic mirror and sharply focused by the objective to form a diffraction limited volume element. A confocal aperture, set in the image plane to reject the out-of-focus light, further reduces the volume from which fluorescence is detected. This is crucial for providing an elliptical observation volume element and enabling submicrometer resolution and quantitative analysis. Following the absorption of energy, fluorescent molecules lose energy through photon emission. Light emitted by fluorescing molecules passing through the confocal volume element (magnified in the insert) is separated from the exciting radiation and the scattered light by a dichroic mirror and barrier filter, and transmitted to the detector. The number of pulses originating from the detected photons, recorded during a specific time interval, corresponds to the measured light intensity. Examples of fluorescence intensity fluctuations recorded in systems with one component undergoing A. free three-dimensional diffusion or B. free three-dimensional diffusion with binding to a surface. Corresponding autocorrelation curves are shown in C and D, respectively. C. Autocorrelation curve G(τ) fitted using equation (2a). The average number of molecules in the observation volume element is determined from the amplitude of the autocorrelation function (1/<i>N</i> = 0.94), and average residence time τ<i>_D</i> from the inflection point. D. Autocorrelation curve G(τ) fitted using equation (2c). The complex shape of the autocorrelation curve indicates that two components with different diffusion times, τ<i>_D1</i> and τ<i>_D2</i>, are present. The average number of particles in the observation volume element is determined from the amplitude of the autocorrelation curve (1/<i>N</i> = 0.8). The ratio of the free fraction <i>versus</i> the bound is given by the ratio of the relative amplitudes (1−<i>f</i> )/<i>f</i>.</p

Effects of ethanol and opioid receptor agonists/antagonists on the MOP surface density in live PC12 cells.

<p>A. Confocal fluorescence images showing subcellular localization of MOP-EGFP (green) in PC12 cells under control conditions (left), upon 3 h treatment with DAMGO (1.0 µM; middle) or naloxone (100 nM; right). EGFP fluorescence was excited using the 488 nm line of the Ar laser. Fluorescence emitted in the range 505–540 nm was collected. B. Schematic drawing of a PC12 cell, showing the location of the observation volume element during FCS measurements. FCS measurements were always performed on the apical side of the plasma membrane. C. Typical autocorrelation curve for MOP-EGFP in not stimulated PC12 cells. FCS measurements were performed and analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004008#s4" target="_blank"><i>Materials and Methods</i></a> section. The dots give the experimental autocorelation curve; the smooth curve is a theoretical autocorrelation curve derived using a two-component model for free 2D-diffusion (eq. 3). Two fractions of MOP-EGFP were identified that could be distinguished by differences in lateral mobility, τ_D1 = (250±150) µs and τ_D2 = (2.5±1.5) ms. The majority of MOP-EGFP was characterized by fast mobility, <i>f</i>₁ = (0.7±0.2). The amplitude of the autocorrelation curve is reciprocally proportional to the average number of MOP-EGFP molecules in the observation volume element (eq. 3). Autocorrelation curves are the basis for the calculation of relative changes in receptor surface densities as in the graph below. D. Relative changes in MOP-EGFP surface density under stimulation with selected drugs: ethanol (stars), naltrexone (diamonds), naloxone (dots), morphine (triangles) and DAMGO (squares). Selective ligands at MOP caused monotonous increase/decrease of MOP-EGFP surface density. Naloxone and naltrexone, acting as antagonists at MOP monotonously increased the MOP-EGFP surface density. The agonists DAMGO and morphine induced rapid internalization of MOP-EGFP, characterized by an internalization half-time <i>t</i>_1/2,agonists = 2.5 min. Ethanol induced an abrupt transient increase in MOP-EGFP surface density, followed by partial internalization of MOP-EGFP, with an apparent internalization half-time of <i>t</i>_1/2,ethanol = 25 min.</p

Normalized temporal autocorrelation curves showing the effect of ethanol and naltrexone on MOP-EGFP lateral mobility in the plasma membrane.

<p>The shift of the autocorrelation curve to shorter correlation times (stars) indicates that ethanol somewhat impels the lateral mobility of MOP-EGFP. In contrast, naltrexone markedly slowed down the lateral mobility of MOP-EGFP. The temporal autocorrelation curve (open circles) assumed a complex shape, indicating the formation of a slowly moving component.</p

Naloxone and naltrexone modulate the effect of ethanol on the plasma membrane lipid dynamics.

<p>Naloxone and naltrexone modulate the effect of ethanol on the plasma membrane lipid dynamics.</p

Corticosterone oscillations during mania induction in the lateral hypothalamic kindled rat—Experimental observations and mathematical modeling

<div><p>Changes in the hypothalamic-pituitary-adrenal (HPA) axis activity constitute a key component of bipolar mania, but the extent and nature of these alterations are not fully understood. We use here the lateral hypothalamic-kindled (LHK) rat model to deliberately induce an acute manic-like episode and measure serum corticosterone concentrations to assess changes in HPA axis activity. A mathematical model is developed to succinctly describe the entwined biochemical transformations that underlay the HPA axis and emulate by numerical simulations the considerable increase in serum corticosterone concentration induced by LHK. Synergistic combination of the LHK rat model and dynamical systems theory allows us to quantitatively characterize changes in HPA axis activity under controlled induction of acute manic-like states and provides a framework to study <i>in silico</i> how the dynamic integration of neurochemical transformations underlying the HPA axis is disrupted in these states.</p></div

Experimentally measured temporal changes in corticosterone concentrations during LHK.

<p>Experimentally measured changes in corticosterone concentrations in individual animals: control (open circle, blue), sham (rectangle, black) and kindled (solid circle, red). The shaded region indicates the interval during which LHK was applied.</p

Effect of LHK duration and intensity on HPA axis activity.

<p><b>A.</b> Changes in HPA axis dynamics induced by LHK of different duration: 50 min, 3 hours, 5 hours, 10 hours and 72 hours (from top to bottom) but the same intensity of a single CRH pulse, 5×10⁻⁸ M. <b>B.</b> Changes in HPA axis dynamics induced by LHK of the same duration (72 hours) but different intensity. The intensity of a single CRH pulse during LHK was: 2×10⁻⁸ M (top), 2×10⁻⁷ M (middle) and 1×10⁻⁶ M (bottom). <b>C.</b> The amplitude of ultradian corticosterone oscillations increases as the intensity of LHK is being increased. Solid circles indicate the highest and the lowest value of the ultradian corticosterone oscillation with the largest amplitude. Open circles indicate the corresponding values under sham conditions, without LHK. <b>D.</b> The frequency of ultradian oscillations increases as the intensity of LHK is being increased. The open circle at the origin indicates the frequency of ultradian oscillations under sham conditions, without LHK.</p

Self-regulation of HPA axis activity.

<p><b>A.</b> Concise schematic presentation of biochemical pathways considered in the reaction model of HPA axis dynamics given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177551#pone.0177551.t001" target="_blank">Table 1</a>. The reaction model includes CRH, ACTH, ALDO and CORT that comprise the backbone of the HPA axis in humans, and CTS the leading glucocorticoid in rodents. Complex interactions between the considered species give rise to positive (+) and negative (-) feedback loops through which the concentration of all reactive species is finely controlled. <b>B.</b> Cholesterol and products of adrenal steroidogenesis included in the reaction model in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177551#pone.0177551.t001" target="_blank">Table 1</a> are shown in black. Intermediates that are presently not included in the reaction model are shown in blue. Steroidogenic enzymes that catalyze specific steps in cholesterol conversion to active steroid hormones are indicated in red.</p

Western blot analysis of protein expression for GSK3β and isoforms of the prolactin receptor.

<p>A) Detection of 80 kDa and 60/70 kDa products with the PRLrI antibody in normal tissues, T47D cells and parathyroid tumours, and 60/70 kDa N-glycosylated PRLr products with the gPRLr antibody in parathyroid tumours. B) Expression of total GSK3β (left) as well as Ser9-phosphorylated GSK3β (right) in parathyroid tumours and normal tissue.</p