Cannabidiol blocks long-lasting behavioral consequences of predator threat stress: Possible involvement of 5HT1A receptors

Posttraumatic stress disorder (PTSD) is an incapacitating syndrome that follows a traumatic experience. Predator exposure promotes long-lasting anxiogenic effect in rodents, an effect related to symptoms found in PTSD patients. Cannabidiol (CBD) is a non-psychotomimetic component of Cannabis sativa with anxiolytic effects. The present study investigated the anti-anxiety actions of CBD administration in a model of PTSD. Male Wistar rats exposed to a predator (cat) received, 1 h later, singled or repeated i.p. administration of vehicle or CBD. Seven days after the stress animals were submitted to the elevated plus maze. To investigate the involvement of 5HT1A receptors in CBD effects animals were pre-treated with WAY100635, a 5HT1A receptor antagonist. To explore possible neurobiological mechanisms involved in these effects, 5HT1A receptor mRNA and BDNF protein expression were measured in the hippocampus, frontal cortex, amygdaloid complex and dorsal periaqueductal gray. Repeated administration of CBD prevented long-lasting anxiogenic effects promoted by a single predator exposure. Pretreatment with WAY100635 attenuated CBD effects. Seven days after predator exposure 5HT1A mRNA expression was up regulated in the frontal cortex and hippocampus. CBD and paroxetine failed to prevent this effect. No change in BDNF expression was found. In conclusion, predator exposure promotes long-lasting up-regulation of 5HT1A receptor gene expression in the hippocampus and frontal cortex. Repeated CBD administration prevents the long-lasting anxiogenic effects observed after predator exposure probably by facilitating 5HT1A receptors neurotransmission. Our results suggest that CBD has beneficial potential for PTSD treatment and that 5HT1A receptors could be a therapeutic target in this disorder. (C) 2012 Elsevier Ltd. All rights reserved.

Alteration in 5HT1A Receptor Activity from a Prenatal Exposure to Dexamethasone in a Stressed and Non-Stressed Adult Male Rat

Synthetic glucocorticoids (GC) are used as a clinical therapeutic to stimulate lung development in fetuses that present the risk of preterm delivery. Previous studies have shown that a prenatal exposure to Dexamethasone (DEX) causes a disturbance in normal GC mediation of neuritic outgrowth, cell signaling, and serotonergic systems. Our hypothesis is that a prenatal exposure to DEX during the third trimester of pregnancy alters 5HT1A receptor function. Pregnant dams were injected daily with 150μg/ml/kg of DEX from gestation day 14 through 19. Control dams were treated with and equal volume of saline. Swim stress followed by elevated plus maze testing was conducted on male rats an hour and a half prior to being sacrificed to induce postnatal acute stress. The non-stressed group was also tested and allowed to return to baseline before sacrifice. Hippocampi were analyzed using a radioligand-receptor binding assay and GTPγS35 incorporation (3H-MPPF antagonist and 8-OH-DPAT agonist, respectively). A significant increase in Kd was found in non-stressed DEX-exposed animals compared to non-stressed controls (p

Development of radiosynthesis methods for 18F-labelled radiopharmaceuticals : General considerations and production of a dopamine transporter radioligand

Positron emission tomography (PET) is a molecular imaging technique that utilises radiopharmaceuticals (radiotracers) labelled with a positron-emitting radionuclide, such as fluorine-18 (18F). Development of a new radiotracer requires an appropriate radiosynthesis method: the most common of which with 18F is nucleophilic substitution with [18F]fluoride ion. The success of the labelling reaction is dependent on various factors such as the reactivity of [18F]fluoride, the structure of the target compound in addition to the chosen solvent. The overall radiosynthesis procedure must be optimised in terms of radiochemical yield and quality of the final product. Therefore, both quantitative and qualitative radioanalytical methods are essential in developing radiosynthesis methods. Furthermore, biological properties of the tracer candidate need to be evaluated by various pre-clinical studies in animal models. In this work, the feasibility of various nucleophilic 18F-fluorination strategies were studied and a labelling method for a novel radiotracer, N-3-[18F]fluoropropyl-2beta-carbomethoxy-3beta-4-fluorophenyl)nortropane ([18F]beta-CFT-FP), was optimised. The effect of solvent was studied by labelling a series of model compounds, 4-(R1-methyl)benzyl R2-benzoates. 18F-Fluorination reactions were carried out both in polar aprotic and protic solvents (tertiary alcohols). Assessment of the 18F-fluorinated products was studied by mass spectrometry (MS) in addition to conventional radiochromatographic methods, using radiosynthesis of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) as a model reaction. Labelling of [18F]beta-CFT-FP was studied using two 18F-fluoroalkylation reagents, [18F]fluoropropyl bromide and [18F]fluoropropyl tosylate, as well as by direct 18F-fluorination of sulfonate ester precursor. Subsequently, the suitability of [18F]beta-CFT-FP for imaging dopamine transporter (DAT) was evaluated by determining its biodistribution in rats. The results showed that protic solvents can be useful co-solvents in aliphatic 18F-fluorinations, especially in the labelling of sulfonate esters. Aromatic 18F-fluorination was not promoted in tert-alcohols. Sensitivity of the ion trap MS was sufficient for the qualitative analysis of the 18F-labelled products; p-[18F]MPPF was identified from the isolated product fraction with a mass-to-charge (m/z) ratio of 435 (i.e. protonated molecule [M+H]+). [18F]beta-CFT-FP was produced most efficiently via [18F]fluoropropyl tosylate, leading to sufficient radiochemical yield and specific radioactivity for PET studies. The ex vivo studies in rats showed fast kinetics as well as the specific uptake of [18F]beta-CFT-FP to the DAT rich brain regions. Thus, it was concluded that [18F]beta-CFT-FP has potential as a radiotracer for imaging DAT by PET.

Neurotoxicidade de pesticidas organofosforados durante o desenvolvimento: alterações bioquímicas e comportamentais

Pesticidas organofosforados são amplamente usados e seu uso constitui um grave problema de saúde pública. A ação clássica destes compostos é a inibição irreversível da acetilcolinesterase, promovendo acúmulo de acetilcolina nas sinapses e hiperestimulação colinérgica. No entanto, as consequências da exposição a baixas doses podem se estender a outros mecanismos de ação e sistemas neurotransmissores. Considerando que crianças constituem um grupo particularmente vulnerável aos efeitos de pesticidas, neste trabalho investigamos os efeitos da exposição aos organofosforados metamidofós (MET) e clorpirifós (CPF) durante o desenvolvimento sobre os sistemas colinérgico e serotoninérgico e sobre o comportamento de camundongos. Para isso, camundongos suíços foram expostos a injeções subcutâneas de MET, clorpirifós ou veículo do terceiro (PN3) ao nono (PN9) dias de vida pós-natal. As doses de exposição foram previamente escolhidas através da construção de uma curva dose-resposta que identificou como mais adequadas para este estudo as doses de 1mg/kg de MET e 3mg/kg de CPF, as quais promoveram em torno de 20% de inibição da acetilcolinesterase. Em PN10, parte dos animais foi sacrificada e foram avaliados os sistemas colinérgico e serotoninérgico no tronco encefálico e córtex cerebral. De PN60 a PN63, os animais foram submetidos a uma bateria de testes comportamentais. Em seguida, estes animais também foram sacrificados tendo sido avaliados os sistemas colinérgico e serotoninérgico. Em PN10, MET e CPF causaram alterações que sugerem aumento da atividade colinérgica respectivamente no tronco e córtex em fêmeas. No sistema serotoninérgico, apenas CPF promoveu alterações, aumentando a ligação ao receptor 5HT1A e transportador 5HT em fêmeas e diminuindo na ligação ao 5HT2. Em PN63, a atividade da acetilcolinesterase foi reestabelecida em todos os grupos. Ainda assim, MET diminuiu a atividade da colina acetiltransferase no córtex e a ligação ao transportador colinérgico no tronco. Quanto aos efeitos do CPF, no tronco, houve redução da atividade da colina acetiltransferase em fêmeas e aumento em machos. Sobre o sistema serotoninérgico, MET e CPF promoveram diminuições no 5HT1A respectivamente no tronco e córtex das fêmeas e CPF aumentou a ligação no córtex de machos. A ligação ao 5HT2 foi aumentada após o tratamento com MET e ao transportador 5HT foi diminuída em fêmeas após o tratamento com clorpirifós. Sobre o comportamento, identificamos comportamento associado à depressão em animais expostos a MET e aumento dos níveis de ansiedade, além de prejuízo de aprendizado/memória após exposição à CPF. Desta forma, nossos resultados indicam que a exposição à metamidofós e clorpirifós durante o desenvolvimento é capaz de alterar, de diferentes formas, a atividade colinérgica e serotoninérgica, mesmo que as doses de exposição sejam toxicologicamente equivalentes. Foram verificados efeitos nas vias neuroquímicas logo após a exposição e após um longo período de interrupção do tratamento, indicando efeitos tardios em sistemas importantes que podem estar associados às alterações comportamentais. Finalmente, o presente estudo reforça a associação epidemiológica entre pesticidas e alterações psiquiátricas e a capacidade da programação de alterações a longo-prazo quando a exposição se dá durante o desenvolvimento.

Endurance training in Wistar rats decreases receptor sensitivity to a serotonin agonist

There is mounting evidence that increased brain serotonin during exercise is associated with the onset of CNS-mediated fatigue. Serotonin receptor sensitivity is likely to be an important determinant of this fatigue. Alterations in brain serotonin receptor sensitivity were examined in Wistar rats throughout 6 weeks of endurance training, running on a treadmill four times a week with two exercise tests per week to exhaustion. Receptor sensitivity was determined indirectly as the reduction in exercise time in response to a dose of a serotonin (1A) agonist, m-chlorophenylpiperazine (m-CPP). The two groups of controls were used to examine (i) the effect of the injection per se on exercise performance and (ii) changes in serotonin receptor sensitivity associated with maturation. In the test group, undrugged exercise performance significantly improved by 47% after 6 weeks of training (4518 ± 729 to 6640 ± 903 s, P=0.01). Drugged exercise performance also increased significantly from week 1 to week 6 (306 ± 69–712 ± 192 s, P = 0.04). Control group results indicated that the dose of m-CPP alone caused fatigue during exercise tests and that maturation was not responsible for any decrease in receptor sensitivity. Improved resistance to the fatiguing effects of the serotonin agonist suggests desensitization of central serotonin receptors, probably the 5-HT1A receptors. Endurance training appears to stimulate an adaptive response to the fatiguing effects of increased brain serotonin, which may enhance endurance exercise performance.

Avaliação comportamental e neuroquímica de ratos em dessincronização forçada: possíveis implicações para um modelo animal de oscilações no humor

Bipolar disorder has been growing in several countries. It is a disease with high mortality and has been responsible by the social isolation of the patients. Bipolar patients have alterations in circadian timing system, showing a phase shift in various physiological variables. There are several arguments demonstrating alterations in circadian rhythms may be part of the bipolar disorder pathophysiology. Given the necessity for further elucidation, the goal of this study was to validate the forced desynchronization protocol as an animal model for bipolar disorder. To do this, Wistar rats were submitted to a forced desynchronization protocol which consists in a symmetrical light dark cycle with 22h. Under this protocol, rats dissociate the locomotor activity rhythm into two components: one synchronized to the light / dark cycle with 22h, and another component with period longer than 24 hours following the animal endogenous period. These rhythms with different periods sometimes there is coincidence, which we named CAP (Coincidence Active Phase) and the opposite phase, non-coincidence, called NCAP (Non-Concidence Active Phase). The hypothesis is that in CAP animals present a mania-like behavior and animals in NCAP depressive-like behavior. We found some evidence described in detail throughout this thesis. In sum, the animals under forced desynchronization protocol were more stressed, showed an increase in stereotypic behaviors such as grooming and reduction in other behaviors such as risk assessment and vertical exploration when compared to the control group. The CAP animals showed increased locomotor activity, especially during the dark phase when compared to controls (rats under T24) and less depressive behavior in the forced swim test. The animals in NCAP showed a higher anxiety in elevated plus maze, but they don t have ahnedonia. The animals under dissociation have more labeled 5HT1A cells at the amygdala area, which appoint that they have more amygdala inhibition. Taking these data together, we could partially validated the forced desynchronization protocol as an animal model for mood oscillations

Synthese, Tritierung, 99m Tc-Markierung und Evaluierung von Benzamid-Derivaten zur Visualisierung der D 2, D 3-Rezeptoren

Die Diagnose und Therapie von neurodegenerativen Krankheiten, wie beispielsweise Morbus Parkinson besitzt in der heutigen Gesellschaft eine immer größere Bedeutung. Über moderne, bild¬gebende nuklear¬medizinische Verfahren wie SPECT und PET ist es mit geeigneten Radioliganden möglich, Morbus Parkinson vor dem Auftreten von Symptomen zu diagnostizieren. Ein wichtiger Ansatzpunkt zur Diagnose von Morbus Parkinson ist die Visualisierung der postsynaptischen Dopamin-Rezeptoren über radioaktiv (11C, 18F, 123I) markierte Benz¬amid-Derivate. Auf Grundlage der (S)-Pyrolidin-2,3-dimethoxy-Benzamid-Struktur des 18F-Liganden Fallypride wurden verschiedene 99mTc-markierte Benzamid-Derivate als potentielle Radio¬liganden zur Parkinson-Diagnostik entwickelt. Um das Potential von Metall-konjugierten Benzamiden abschätzen zu können, wurden zunächst einfache Vergleichssubstanzen entwickelt. Diese sollten die Einführung eines Chelators simulieren und wurden hierfür hinsichtlich ihrer in vitro-Bindungsaffinitäten zu den Dopamin-, Serotonin- und adrenergen Rezeptoren evaluiert. Die zunächst entwickelten Derivate mit unterschiedlichen Kettenlängen zur Kopplung des Chelators zeigten für die Propylkette Affinitäten im nanomolaren Bereich. Im Anschluss sollten basierend auf diesen Ergebnissen vier verschiedene Chelatoren (Carbony-Cyclopentadienyl, Amido-Cyclopentadienyl, 2-Pyridyl-Imin und N2S2) über eine Propylkette an die 5-Position der Benzamidgrundstruktur gekoppelt werden. Die geplante Synthese des Carbonyl-Cyclopentadienyl-Derivates gelang jedoch nicht. Für die weiteren Chelatoren (Amido-Cyclopentadieny, 2-Pyridyl-Imin und N2S2) konnten die jeweiligen Markierungs¬vorläufer und Rhenium-Komplexe dargestellt werden, die ebenfalls hinsichtlich ihrer Bindungs¬affinitäten evaluiert wurden. Die erzielten Affinitäten zeigten, dass eine Über¬tragung der Affinitäten der einfachen Vergleichssubstanzen auf die komplexeren Metall-Benzamide nicht möglich war. Insbesondere der N2S2-Rhenium-Komplex besitzt nur noch geringe Affinität (490 - 900 nM) zu den D2- und D3-Rezeptoren. Die mittel-affinen 2-Pyridyl-Imin- und Amdio¬cyclopentadien-Komplexe wurden mit 99mTc markiert und die Markierungsausbeute hinsichtlich Reaktionstemperatur, Markierungs-vorläuferkonzentration und Heizmethoden optimiert. Dabei konnte der Imin-Komplex quantitativ mittels fac-[99mTc(CO)3(H2O)3]+ in 30 Minuten bei 45°C markiert werden. Der Amido-Cyclopentadien-Komplex konnte über die Umsetzung des Ferrocen-Markierungsvorläufer mit Mn(CO)5Br und [99mTcO4]- in Ausbeuten von bis zu 60 % markiert werden. Im Anschluss an die Markierungen wurden die 99mTc-Komplexe über HPLC isoliert und in in vitro-Auto¬radiographien von Rattenhirnschnitten weiter evaluiert. Die erhaltenen Ergebnisse bestätigten die für die Rhenium-Komplexe erzielten Affinitäten und zeigten keine spezifische Anreicherung in bestimmten Hirnarealen. Aus diesen Ergebnissen kann ge¬schlossen werden, dass die dargestellten 99mTc-Benzamide aufgrund mangelnder Affinitäten und einer hohen unspezifischen Bindung keine geeigneten Liganden zur Darstellung der D2- und D3- Rezeptoren sind. Um die dargestellten 99mTc-Benzamide mit [18F]Fallypride vergleichen zu können, wurde zusätzlich [3H]Fallypride dargestellt. Hierfür wurde zunächst der Nor-Markierungsvor¬läufer synthetisiert und die Markierungsausbeute optimiert. Die finale Umsetzung mit [3H]Methylnosylat ergab nach HPLC-Aufreinigung 15 mCi [3H]Fallypride mit einer radio¬chemischen Reinheit von >99,5 %. Erste Autoradiographien zeigten eine hohe Anreicherung des Liganden im Striatum, verbunden mit einer sehr niedrigen unspezifischen Bindung.

EFFECTS OF PRENATAL DEXAMETHASONE ON HIPOPCAMPAL SEROTONIN 1A RECEPTORS IN ADULT MALE RATS

The main activation route for the stress response is the hypothalamo-pituitaryadrenal axis (HPA) and the sympatho-adrenomedullary system. The HPA axis is a neuroendocrine feedback loop mediated by an array of tissue specific hormones, receptors and neurotransmitters that regulate glucocorticoid (GC) release. GCs are steroidal hormones produced by the adrenal glands and are key players in a negativefeedback loop controlling HPA activity. They influence the HPA axis through glucocorticoid receptors in the hypothalamus and pituitary and through both glucocorticoid (GR) and mineralcorticoid receptors (MR) that are co-localized in the hippocampus. Repeated or chronic stress exerts a negative influence on these HPA axis regulatory sites and contributes to potentially pathological conditions, especially during early development. For example, chronic stress promotes increased maternal adrenal gland secretion of glucocortiocoid, leading to abnormally high concentrations of GC inthe fetal environment. The timing and maturation of the HPA axis relative to birth is highly species specific and is closely linked to landmarks in fetal development. In rats this development of the HPA axis takes place in utero and continues even shortly after birth. It is likely that the maternal endocrine environment will affect fetal development during this critical time point and may alter the overall set point for the expression ofgenes and their protein products that mediate fetal HPA axis function. Dexamethasone (DEX) is a synthetic glucocorticoid (sGC) and is a consensus treatment in preterm pregnancies used to expedite fetal lung development. However it has been shown that DEX causes long term physiological and behavioral disorders in prenatally-exposed laboratory animals. Previous studies have also shown that it alters the MR: GR receptor ratio in the hippocampus. Taking into consideration corticosteroid regulation of serotonin receptors, especially 5HT1A receptors and their putative interaction with glucocorticoid receptors in the hippocampus, we hypothesized that prenatal DEX exposure would lead to changes in the expression and function of 5HT1A receptors in the hippocampus. We administered DEX to rat dams during the last trimester of gestation and investigated the changes in these receptors in the adult rat offspring. Radioligand receptor binding assays were used to study hippocampal 5HT1A receptor binding affinity and number. Our results demonstrate that hippocampal 5HT1A receptors are increased in the DEX animalscompared with controls by 36%, with no change in binding affinity. The efficiency of ligand-induced receptor signal transduction via G-protein activation was also studied using [35S]GTPγS incorporation assay. Using this technique, we showed that there was no significant difference in the maximum ligand mediated stimulation (Emax) of 5HT1Areceptors between control and dex exposed animals. However, the intracellular signalling efficiency of hippocampal 5HT1A receptors was diminished, since a significant increase in EC50 values was obtained with the dex exposed group showing a value 51% higherEC50 than controls. Taken together these data illustrate a considerable change in the 5HT1A component of the serotonergic system following prenatal DEX exposure.

Galanin N-Terminal fragment (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT in the Forced Swimming Test.

Galanin and Galanin (1-15) [GAL(1-15)] are implicated in anxiety- and depression related behaviors. Moreover, Galanin modulates 5-HT1A receptor (5-HT1AR) function at autorreceptor and postsynaptic level in the brain. In this study, we have analysed the ability of GAL(1-15) to modulate the effects of the 8-OH-DPAT agonist in the Forced Swimming Test (FST). Groups of rats were assessed in the FST. In the first set of experiments, to evaluate the interactions of 8-OH-DPAT and GAL(1-15), rats received subcutaneously (s.c) the effective doses of 8-OH-DPAT (0.25mg/Kg) 60min before the test and intracerebroventricularly (icv) GAL(1-15)1nmol 15min before the tests alone or in combination. In the second set of experiments, groups of rats received s.c. 8-OH-DPAT (0.125mg/Kg), icv GAL(1-15) 1nmol and icv the GALR2 antagonist M871 3 nmol alone or in combination. The locomotor activity was analysed in the open field test. GAL(1-15) 1nmol enhanced the antidepressant-like effects mediated by the effective dose of the 8-OH-DPAT. GAL(1-15) significantly decreased the immobility (p<0.05) and climbing (p<0.05) and increased the swimming (p<0.01) behaviour induced by an effective dose of 8-OH-DPAT (0.25mg/Kg) in FST. Moreover, after coadministration of GAL(1-15) and threshold dose of 8-OH-DPAT (0.125mg/Kg) a significant decreased appeared in immobility (p<0.01) and climbing (p<0.01) and increased the swimming behavior (p<0.001) vs 8-OH-DPAT group. Moreover, M871 blocked completely this interaction. These results indicate that GAL(1-15) enhances the antidepressant effects induced by 8-OH-DPAT in the FST. These findings may give the basis for the development of novel therapeutic drugs. This study was supported by Junta de Andalucía CVI6476.