Administration of URB597, Oleoylethanolamide or Palmitoylethanolamide Increases Waking and Dopamine in Rats

-acylethanolamines or acylethanolamides. The hydrolysis of OEA and PEA is catalyzed by the fatty acid amide hydrolase (FAAH). A number of FAAH inhibitors that increase the levels of OEA and PEA in the brain have been developed, including URB597. In the present report, we examined whether URB597, OEA or PEA injected into wake-related brain areas, such as lateral hypothalamus (LH) or dorsal raphe nuclei (DRN) would promote wakefulness (W) in rats.Male Wistar rats (250–300 g) were implanted for sleep studies with electrodes to record the electroencephalogram and electromyogram as well as a cannulae aimed either into LH or into DRN. Sleep stages were scored to determine W, slow wave sleep (SWS) and rapid eye movement sleep (REMS). Power spectra bands underly neurophysiological mechanisms of the sleep-wake cycle and provide information about quality rather than quantity of sleep, thus fast Fourier transformation analysis was collected after the pharmacological trials for alpha (for W; α = 8–12 Hz), delta (for SWS; δ = 0.5–4.0 Hz) and theta (for REMS; θ = 6.0–12.0 Hz). Finally, microdialysis samples were collected from a cannula placed into the nucleus accumbens (AcbC) and the levels of dopamine (DA) were determined by HPLC means after the injection of URB597, OEA or PEA. We found that microinjection of compounds (10, 20, 30 µg/1 µL; each) into LH or DRN during the lights-on period increased W and decreased SWS as well as REMS and enhanced DA extracellular levels.URB597, OEA or PEA promoted waking and enhanced DA if injected into LH or DRN. The wake-promoting effects of these compounds could be linked with the enhancement in levels of DA and indirectly mediated by anandamide

Cannabidiol, a constituent of Cannabis sativa, modulates sleep in rats

AbstractΔ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are two major constituents of Cannabis sativa. Δ9-THC modulates sleep, but no clear evidence on the role of CBD is available. In order to determine the effects of CBD on sleep, it was administered intracerebroventricular (icv) in a dose of 10μg/5μl at the beginning of either the lights-on or the lights-off period. We found that CBD administered during the lights-on period increased wakefulness (W) and decreased rapid eye movement sleep (REMS). No changes on sleep were observed during the dark phase. Icv injections of CBD (10μg/5μl) induced an enhancement of c-Fos expression in waking-related brain areas such as hypothalamus and dorsal raphe nucleus (DRD). Microdialysis in unanesthetized rats was carried out to characterize the effects of icv administration of CBD (10μg/5μl) on extracellular levels of dopamine (DA) within the nucleus accumbens. CBD induced an increase in DA release. Finally, in order to test if the waking properties of CBD could be blocked by the sleep-inducing endocannabinoid anandamide (ANA), animals received ANA (10μg/2.5μl, icv) followed 15min later by CBD (10μg/2.5μl). Results showed that the waking properties of CBD were not blocked by ANA. In conclusion, we found that CBD modulates waking via activation of neurons in the hypothalamus and DRD. Both regions are apparently involved in the generation of alertness. Also, CBD increases DA levels as measured by microdialysis and HPLC procedures. Since CBD induces alertness, it might be of therapeutic value in sleep disorders such as excessive somnolence

Intrastriatal Grafting of Chromospheres: Survival and Functional Effects in the 6-OHDA Rat Model of Parkinson's Disease

<div><p>Cell replacement therapy in Parkinson’s disease (PD) aims at re-establishing dopamine neurotransmission in the striatum by grafting dopamine-releasing cells. Chromaffin cell (CC) grafts produce some transitory improvements of functional motor deficits in PD animal models, and have the advantage of allowing autologous transplantation. However, CC grafts have exhibited low survival, poor functional effects and dopamine release compared to other cell types. Recently, chromaffin progenitor-like cells were isolated from bovine and human adult adrenal medulla. Under low-attachment conditions, these cells aggregate and grow as spheres, named chromospheres. Here, we found that bovine-derived chromosphere-cell cultures exhibit a greater fraction of cells with a dopaminergic phenotype and higher dopamine release than CC. Chromospheres grafted in a rat model of PD survived in 57% of the total grafted animals. Behavioral tests showed that surviving chromosphere cells induce a reduction in motor alterations for at least 3 months after grafting. Finally, we found that compared with CC, chromosphere grafts survive more and produce more robust and consistent motor improvements. However, further experiments would be necessary to determine whether the functional benefits induced by chromosphere grafts can be improved, and also to elucidate the mechanisms underlying the functional effects of the grafts.</p></div

Survival and motor efficacy of chromosphere cell grafts.

<p>Survival and motor efficacy of chromosphere cell grafts.</p

Estimation of chromosphere graft survival.

<p>(<b>a</b>) One out of four of the total coronal sections of the striatum (represented by dotted squares) were selected for immunofluorescence staining for TH. The antero-posterior extension of the graft along the striatum was estimated by determining the number of coronal sections of the striatum in which grafted cells could still be visualized, as schematized in the figure. As time after grafting progressed, the number of slices with grafted cells became smaller, indicating an antero-posterior shrinking of the grafted area due to cell death. (<b>b</b>) Quantification of the TH⁺ area of a graft. TH⁺ immunofluorescence reconstructions of the striatum (left panel) at 10x magnification were transformed into 8-bit images (middle panel), followed by binary transformation (right panel) to easily quantify the total area of the graft in each striatal section using the area quantification option in ImageJ. The insert framed by a dotted white square in the middle panel highlights the region containing the graft over which we quantified the TH⁺ area. (<b>c</b>) Graph of the estimated TH⁺ area <i>vs</i> the number of TH⁺ cells counted directly. Each data point represents the number of cells or the TH⁺ area determined for a single coronal section. Data points from grafts evaluated at 1, 2, 4 or 12 wpg are shown in different colors. The solid line is the linear regression to the data, with parameters shown at the bottom right. The dotted lines denote the uncertainty of the linear fit. (<b>d</b>) Comparison of the estimation (denoted by letter E in the graph) of graft total cell number using the extrapolation of measured TH⁺ area to the linear regression in (<b>c</b>) with the total number of TH⁺ cells directly counted (denoted by C in the graph) shows that the approximate method yields very similar results to the direct method with no statistical differences (multivariate ANOVA, P < 0.05, F = 0.0043, p = 0.9480).</p

Comparison between chromosphere grafts and CC grafts.

<p>(<b>a</b>) Percentage of change in circling behavior induced by amphetamine in 6-OHDA lesioned animals with either chromosphere (n = 7, orange) or CC (n = 8, purple) grafts and in a 6-OHDA lesioned group (n = 6). Each bar represents the mean ± SEM. The dotted horizontal line denotes a reduction in turn number of 50%. Significant differences in chromaffin grafted animals were observed at 2 and 8 wpg when compared to the lesioned group without graft (repeated measures multivariate ANOVA, P < 0.05, F = 6.991, DF = 23, p < 0.0001; followed by Tukey´s multiple comparisons <i>post hoc</i> test, p < 0.05*, p < 0.01** and p < 0.001***) (black asterisks), but not when compared with their circling behavior before grafting (repeated measures ANOVA, F = 1.142, r² = 0.1402, p = 0.3485). (<b>b</b>) The survival of chromaffin grafted cells was determined by directly counting TH⁺ cells in immunostained coronal brain slices. The dots represent the number of surviving grafted cells from single animals, and the bars denote the mean ± SEM. The dotted horizontal line represents the number of cells equivalent to 1 x 10³. A significant difference was observed between chromaffin and chromosphere cell survival (Unpaired t-test, P > 0.05, t = 3.572, df = 10, p = 0.0051**). (<b>c</b>) Percentage of dopaminergic cells in the lesioned SNpc in relation to the non-lesioned hemisphere in animals unilaterally lesioned with 6OHDA and grafted with either CC (grey) or chromospheres (black). The number of TH⁺ cells in the SNpc of both the lesioned and non-lesioned sides was determined by direct cell counting of TH immunostained brain slices. Data was normalized to the number of counted TH⁺ cells in the non-lesioned SNpc. Each data point represents an individual animal, and the mean ± SEM is indicated by solid horizontal lines. The dotted horizontal line denotes a loss of 20% of TH⁺ cells in the lesioned hemisphere compared to the non-lesioned SNpc. No significant differences were observed (two-tailed unpaired t-test) between animals grafted with either chromospheres or CC, suggesting that the degree of lesion induced by the 6-OHDA toxin was similar in all the analyzed animals.</p

<i>In vitro</i> analysis of noradrenaline, adrenaline and dopamine release in chromospheres and CC.

<p>(<b>a-b</b>) Adrenaline, noradrenaline and dopamine release under basal conditions was determined by HPLC-ED in 3- and 9- days cultured-CC and 9 days cultured-chromospheres. 3 days CC and 9 days chromospheres were obtained from 8 independently processed adrenal glands, whereas 9 days CC were obtained from 3 independently processed adrenal glands. Data collection and analysis from cells obtained from different glands was also done separately. The extracellular medium was recollected for quantification of released catecholamines. Catecholamine quantities are expressed in nmol. (<b>a</b>) Significant differences were observed for adrenaline (3-days CC: 0.9726 ± 0.3828 nmol; 9-days CC: 4.057 ± 0.7481 nmol; 9-days chromospheres: 1.065 ± 0.2261 nmol; multivariate ANOVA, P > 0.05, F = 17.36, DF = 2, p < 0.0001; followed by Tukey´s multiple comparisons <i>post hoc</i> test, p < 0.0001****) and (<b>b</b>) dopamine (3 days CC: 0.0058 ± 0.0038 nmol; 9 days CC: 0.087 ± 0.043 nmol; 9 days chromospheres: 0.12 ± 0.019 nmol; ANOVA, P > 0.05, F = 12.53, r² = 0.6103, p = 0.0005; followed by Tukey´s multiple comparisons <i>post hoc</i> test, p = 0.0145* and p = 0.0004***). (<b>c</b>) Graphic representation of the percentage of dopamine released relative to the total catecholamines released (adrenaline + noradrenaline + dopamine). Significant differences were observed between 9-days chromospheres compared to 9 and 3 days CC (ANOVA, P > 0.05, F = 27.42, r² = 0.7231, p < 0.0001; followed by Tukey´s multiple comparisons <i>post hoc</i> test, p < 0.0001****).</p

Relation between chromosphere survival and motor improvement.

<p>Amphetamine-induced circling behavior was used to determine whether there is a relation between chromosphere survival and reduction in turn number in grafted animals. (<b>a</b>) Percentage of change in amphetamine induced circling behavior in 6-OHDA-lesioned animals grafted with chromospheres. Each animal is represented by a different dot color. The percentage of change was calculated using the initial number of turns for each 6-OHDA-treated animal before grafting as reference, so that negative values denote a decrease in the number of turns, whereas positive values denote an increase. The dotted horizontal lines denote a reduction in turn number of 50% and 100%. At the end of the experiment 6 out of 13 animals reduced their number of turns by ≥ 10%. Animals were sacrificed after the last evaluation and the number of surviving grafted cells were determined by direct counting. (<b>b</b>) Relation between the percentage of change in turn number at 4 wpg relative to the number of turns before grafting (x-axis) and the number of estimated surviving cells at 4 wpg (y-axis) for chromosphere-grafted animals used for the behavioral experiments in (<b>a</b>). The graph quadrant corresponding to a reduction in turn number is highlighted in teal. Each dot represents data from a single animal. The obtained relation shows that all animals with a reduction in turn number have surviving grafted cells, and that, with two notable exceptions, animals without changes in turn number did not present any surviving grafted cells.</p

EEG alpha (for W; α = 8–12 Hz. Panel A), delta (for SWS; δ = 0.5–4.0 Hz. Panel B) and theta (for REMS; θ = 6.0–12.0 Hz. Panel C) power spectra (units, µV²) in rats after the following treatments into lateral hypothalamus: Sham, vehicle, URB597, OEA or PEA (30 µg/1 µL; each compound).

<p>Data were calculated over 3 consecutive hours at every time point (Mean ± SEM; * vs. Sham/Vehicle, <i>p</i><0.05).</p