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No description available.

Cocaine is a powerfully active psychostimulant which exerts its effects through blockade of dopamine, serotonin and norepinephrine transporters and resultant increases in extracellular levels of these neurotransmitters. Much of the focus on cocaine abuse in the literature has been directed towards study of the dopamine system; however, several studies have identified a role for the serotonin system in regulating the rewarding effects of cocaine as well. Specifically, the serotonin 2C (5-HT2C) receptor regulates cocaine-induced alterations in serotonin and dopamine levels in an inhibitory manner, and 5-HT2C receptor agonist treatment attenuates cocaine-induced behaviors like self-administration. In the first aspect of the current thesis study, the effects of activation of 5-HT2C receptors on cocaine-induced conditioned place preference and behavioral sensitization were assessed. It was found that pretreatment with a 5-HT2C receptor agonist, Ro 60-0175, on cocaine (10 mg/kg) conditioning days of the conditioned place preference paradigm, attenuated the development of conditioned place preference in a dose-dependent manner. These results suggest that activation of 5-HT2C receptors inhibits the euphoric effects elicited by cocaine. Behavioral sensitization studies demonstrated that pretreatment with Ro 60-0175 prior to cocaine (10 mg/kg) over a 5 day period attenuated cocaine-induced hyperactivity. When injected with a cocaine challenge injection 10 days after the last cocaine injection, mice pretreated with Ro 60-0175 demonstrated lower levels of locomotor activity as compared to saline pretreated, cocaine-injected mice. This portion of the first study demonstrated that 5-HT2C receptor activity attenuated acute cocaine-induced conditioned reward, hyperactivity and the development of long-term alterations of cocaine exposure, as measured by behavioral sensitization. The second aspect of the current study focused on the regulation of anxiety produced by withdrawal from chronic cocaine administration. Anxiety during cocaine withdrawal is a component of the negative affective state often experienced by cocaine-dependent individuals during abstinence from drug use. Anxiety during cocaine withdrawal is likely to increase an individual's susceptibility to relapse to drug use in alleviation of this negative symptom. Studies have shown a downregulation of the serotonin and dopamine systems during withdrawal that potentially contributes to anxiety symptoms. As the 5-HT2C receptor exerts inhibitory control over both the serotonin and dopamine systems, it was hypothesized that blockade of 5-HT2C receptors would attenuate anxiety-like behavior during cocaine withdrawal. Previous studies have identified co-localization of 5-HT2C receptors on inhibitory gamma-aminobutyric acid (GABA) neurons, thus it was hypothesized that a 5-HT2C receptor-GABA mediated mechanism would be involved in the regulation of anxiety during withdrawal. The dorsal raphe brain region was targeted in these studies, as this region is the primary source of serotonin for forebrain structures. The actions of cocaine on the serotonin system likely originate with influence of cocaine on the dorsal raphe neurocircuitry, with implications for dysregulation in downstream projection areas of the dorsal raphe. In this portion of the current thesis study, electrophysiology techniques were used to measure GABA activity in subregions of the dorsal raphe either 30 minutes, 25 hours, or 7 days following a 10-day chronic binge cocaine paradigm (15 mg/kg, 3 injections per day at 1 hour intervals). Controls received saline injections. Mice were tested for anxiety-like behavior on the elevated plus maze and then brain slices were collected for electrophysiology recordings. It was found that at 25 hours of withdrawal, cocaine-treated mice demonstrated heightened anxiety-like behavior on the elevated plus maze, as compared to saline controls. Mice tested during an active cocaine stage 30 minutes after the last injection, or at 7 days of withdrawal did not demonstrate increased anxiety-like behavior. Heightened GABA activity was exhibited in serotonin cells from cocaine-withdrawn mice at 25 hours of withdrawal, an effect that was normalized upon 5-HT2C receptor blockade. No differences were observed at 30 minutes after the last cocaine injection; however, there was an anatomical shift observed at 7 days of withdrawal, in that heightened GABA activity exhibited in two subregions of the dorsal raphe (dorsomedial and ventromedial aspects) at 25 hours of withdrawal shifted to the lateral wing areas at 7 days of withdrawal. The differential regulation of the three subregions has implications on serotonin output to projection areas and contribution to anxiety mechanisms. It was found that systemic administration and local intra-dorsal raphe administration of the 5-HT2C receptor antagonist, SB 242084, prior to elevated plus maze testing attenuated anxiety-like behavior in cocaine-withdrawn mice at 25 hours of withdrawal. Taken together, this portion of the thesis study demonstrated that 5-HT2C receptor activity, specifically within the dorsal raphe, regulates anxiety during cocaine withdrawal, through influence on the GABA inhibitory feedback system. A final aspect of the current thesis study addressed the link between dorsal raphe 5-HT2C receptor activity and activity at downstream structures in the context of cocaine withdrawal-induced anxiety, particularly the nucleus accumbens. Since the dorsal raphe is important in providing serotonin input for brain regions largely involved in regulating the effects elicited by cocaine, it is likely that dysregulation of dorsal raphe signaling during withdrawal has influence on the regulation of downstream structures in the contribution of anxiety mechanisms produced by cocaine withdrawal. It was found that dorsal raphe 5-HT2C receptor blockade attenuated cocaine withdrawal-induced reductions in cFos immunoreactivity in the nucleus accumbens. Further work is needed to investigate these interactions in the context of cocaine withdrawal-induced anxiety. Lastly, autoradiography experiments assessed the effects of cocaine withdrawal-induced anxiety on 5-HT2C receptor expression in various brain regions including the dorsal raphe, medial prefrontal cortex, nucleus accumbens, caudate putamen, and ventral tegmental area. A significant decrease in 5-HT2C receptor binding was found in the dmDR region of cocaine-withdrawn mice as compared to saline controls; however, no differences were found between groups in other regions. Future studies testing 5-HT2C receptor signaling are needed to fully understand the impact of cocaine withdrawal-induced anxiety on receptor function in these structures. In conclusion, the first portion of the current study showed that activation of 5-HT2C receptors attenuated the rewarding and locomotor sensitizing effects of cocaine, as evidenced by conditioned place preference and behavioral sensitization studies. In the second aspect of the current thesis study, we have established a role for the 5-HT2C receptor in the regulation of anxiety during cocaine withdrawal. During withdrawal, blockade of 5-HT2C receptor activity, both global as well as local dorsal raphe blockade, attenuated anxiety at 25 hours of withdrawal. This attenuation of cocaine withdrawal-induced anxiety resultant of 5-HT2C receptor blockade was likely due to a suppression of increased GABA activity evident in serotonin cells from cocaine-withdrawn mice. / Pharmacology

The essential amino acid tryptophan serves as the precursor to serotonin, a neurotransmitter which regulates hunger and satiety, promotes sleep, relieves depression, and is found in elevated levels following exercise. A quantitative deficit in serotonin or abnormality in its mode of action is thought to be one cause behind several psychological disorders such as depression, eating disorders, and obsessive-compulsive disorder. A decrease in dietary tryptophan has been shown to decrease serotonin production thereby, theoretically, producing the effects of a hyposerotonergic state. The purpose of this study was to further elucidate serotonin’s role in influencing an individual’s activity level by inducing a decrease in this neurotransmitter via a tryptophan-depleted diet. Specifically, 40 individually-housed mice were divided into four groups of ten. Group C received a control diet while group CW consumed a control diet and had access to a running wheel. Group E received a diet low in tryptophan while group EW was given the experimental diet and had access to a running wheel. A seven-day adaptation period was followed by a ten-day experimental period during which time groups E and EW received the experimental diet. Only the food consumption of group EW increased significantly over time. Running wheel activity was monitored every 12 hours corresponding to a light/dark cycle. An analysis of this data revealed a significant post-test rise in the activity levels of the mice in group EW compared to the activity of group CW. The wheel revolutions per 24 hours dropped by 17% in group CW while group EW’s revolutions increased by 19%. Furthermore, while revolutions increased from pre-test to post-test in only four out of 10 control subjects, they increased in all nine of the experimental subjects. It seems likely that the tryptophan depletion caused a decrease in brain serotonin levels. Therefore, the results of this experiment support the theory that the mice on a low-tryptophan diet increased their activity in a subconscious effort to normalize serotonin levels which would bring about a return of the desirable effects of this neurotransmitter. / Master of Science

Die Heterodimerisierung von G- Protein gekoppelten Rezeptoren (GPCR) stellt ein aktuelles Forschungsgebiet dar, das molekulare Erklärungsmöglichkeiten für die Vielfalt der Signalwege über solche Rezeptoren aufzeigt. Die genauen Funktionen diese Konstrukte in vivo sind bisher erst in Ansätzen erforscht, ebenso wenig die molekularbiologischen Mechanismen. Für die beiden Serotoninrezeptoren 5-HT1A und 5-HT7 konnte Heterodimerisierung molekular nachgewiesen werden, in ihren physiologischen Mechanismen und Effekten sollte daher eine Charakterisierung vorgenommen werden. Mittels elektrophysiologischer Messverfahren wurden Ströme an dem heterologen Expressionsmodell der Oozyten des Krallenfrosches Xenopus laevis mittels Voltage-Clamp Technik an Kaliumionenkanälen (Kir3 und TASK-1) gemessen. Hierbei konnte gezeigt werden, dass die heterodimere Koexpression beider Rezeptoren eine signifikante Reduktion des Rezeptor-aktivierten Kanalstroms im Vergleich zur homomeren Expression zur Folge hatte. Weitere Experimente konnten dann zeigen, dass diese Effekte spezifisch für dieses Rezeptorheterodimer sind, und dass die Effekte von der Dosis bzw. dem Verhältnis der exprimierten cRNA abhängen. In Fluoreszenzmessung konnte zudem gezeigt werden, dass die Reduktion der Stromamplitude in der heterodimeren Expression nicht auf eine Reduktion von Kanalproteinen in der Zellmembran zurückzuführen ist. Zur weiteren Charakterisierung des bisher erst in Ansätzen erforschten 5-HT7 Rezeptors wurde dieser abschließend mit einem ß- adrenergen Rezeptor verglichen, der über den gleichen Signalweg bzw. Ionenkanal funktioniert. Auch hier zeigte sich eine signifikante Reduktion des Kanalstroms beim 5-HT7 Rezeptor. Die physiologische Relevanz dieser Ergebnisse liegt darin begründet, dass ein weiterführendes Verständnis von 5-HT Rezeptor vermittelten Signalwegen, insbesondere von der Bedeutung und den Mechanismen ihrer Heterodimersierung, neue pathophysiologische Zusammenhänge verdeutlicht. Speziell im Hinblick auf Erkrankungen, die mit den 5-HT Rezeptoren assoziiert sind, wie etwa Depressionen und Angststörungen, soll sich hieraus die Möglichkeit spezifischerer Therapien ergeben. / Heteromerisation of G-protein coupled receptors (GPCR)is a current object of research to find out diversity of signaling pathways. The functional details of those constructs in vivo are not yet understood, also molecular mechanisms. For the Serotonin receptors 1A and 7 heteromerisation recently could be shown, therefore intention now is to make a physiological characterization. By electrophysiological methods, i.e. voltage clamp, currents of potassium channels (Kir3 and TASK-1) could be detected using the heterologous expression system of oocytes of xenopus leavis. So we could demonstrate, that heterologous expression of both receptors leads to a reduced current amplitude in comparison to homologous expression of one receptor. Those effects where shown to be specific and dependend of cRNA dose. By using fluorescense tagged Kir-channel we could demonstrate that the effect doesn't base on less channel protein in cell surface of the oocytes. Another point of interest was the characterization of Serotonin-7 channel. Therefore we analyzed a dose-response-relationship, afterwards we compared data with a ß-adrenergic receptore in heteromeric expression with Serotonin-1A. The physiological relevance of those experiments is to understand serotonin pathways an metabolism that is very important in development of mental disorders of fear or major depression.

No description available.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Background: Selective serotonin reuptake inhibitor (SSRI) antidepressants prevent serotonin (5-HT) uptake by the serotonin transporter (SERT). Since blood platelets express SERT, SSRIs may modify platelet function and the risk of cardiovascular disease. However, the beneficial or adverse effects of SSRIs on arterial thrombosis are poorly characterised and detailed in vitro experimental data is limited. The SSRI citalopram is a racemate, the (S)-isomer being the more potent SERT inhibitor. Although citalopram has been shown to inhibit platelets in vitro, it is unclear whether this is mediated via SERT blockade. Aim: To determine if citalopram inhibits platelet function via SERT blockade, or through a novel mechanism of action. Findings: 5-HT uptake into platelets was blocked by both citalopram isomers at concentrations that had no apparent effect on platelet function. Despite the (S)-citalopram isomer being the more potent SERT inhibitor, (R)-citalopram was equally potent at inhibiting other platelet functions. These findings strongly suggest that inhibition of platelet function by citalopram in vitro is not mediated by blocking SERT. Subsequent experiments identified two putative mechanisms for citalopram-mediated platelet inhibition: 1) citalopram did not inhibit calcium store release induced by the platelet agonist U46619, despite blocking subsequent Rap1 activation. A credible target for this inhibitory mechanism is the calcium and diacylglycerol guanine nucleotide exchange factor-1 (CalDAG-GEFI): 2) citalopram suppressed early protein phosphorylation within the GPVI pathway, resulting in the inhibition of subsequent platelet responses. Further experiments show that other commonly used antidepressants also inhibit platelets. As with citalopram, inhibition was only observed at concentrations above those required to block SERT, suggesting that alternative inhibitory mechanism(s) are responsible. Conclusions: Data presented in this thesis support two novel putative mechanisms of citalopram-induced platelet inhibition. These findings demonstrate that citalopram and other antidepressants inhibit platelets independently of their ability to block SERT-dependent 5-HT transport. The identification of thesemechanisms provides a pharmacological approach to develop novel antiplatelet agents based on current antidepressants.

In the present study non-5-HT₁(A)/non-5-HT₁(C) binding sites in the rabbit caudate nucleus (CN) were examined to determine if they might be identical to the recently discovered 5-HT₁(D) sites in the bovine CN. The characterizations were carried out measuring high-affinity [³H]5-HT binding under conditions where 5-HT₁(A) and 5-HT₁(C) sites were pharmacologically masked in both tissues. Comparison of the pharmacologic profiles of the bovine 5-HT₁(D) and rabbit non-5-HT₁(A)/non-5-HT₁(C) sites revealed similarities, but showed distinct differences. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by GTP than was [³H]5-HT binding in the rabbit CN, and this effect was differentially sensitive to calcium and other divalent cations (i.e., Mg²⁺, Mn²+)⁺in the two tissues. [³H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by NEM than it was in the rabbit CN. Thus, it may be concluded that the non-5-HT₁(A)/non-5-HT₁(C) [³H]5-HT binding sites in rabbit CN are distinct from those in the bovine CN, and we propose that they be tentatively identified as 5-HT₁(R) to distinguish them from the 5-HT₁(D) site.

The crayfish claw opener neuromuscular junction (NMJ) is a biological model for studying presynaptic neuromodulation by serotonin and synaptic vesicle recycling. Serotonin acts on crayfish axon terminals to increase the release of the neurotransmitter glutamate, but a complete understanding of its mechanisms of action are unknown. In order to sustain enhanced neurotransmission over long periods of time, it was hypothesized that serotonin recruits (activates) a population of previously non-recycling vesicles to become releasable and contribute to neurotransmission. To determine if serotonin activates a distinct population of synaptic vesicles, FM1-43 fluorescence unloading experiments were performed on crayfish excitatory opener axon terminals. These experiments could not resolve a serotonin-activated population of synaptic vesicles, but instead revealed that synaptic vesicles change behaviour in axon terminals independent of serotonin, with vesicles becoming less likely to exocytose and unload FM1-43 dye over time. The change in behaviour was hypothesized to be due to conversion of vesicles from a recycling (releasable) status to a reserve (reluctant to release) status. Synaptic vesicle pool localization was then tested using photoconversion of FM1-43 and transmission electron microscopy techniques. The spatial location of FM1-43-labeled vesicles fixed 2 minutes following 20 Hz stimulation did not reveal retention of vesicles specifically near release sites and the distribution of FM1-43-labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 seconds following stimulation, however, contained numerous endosome-like structures - the most frequently observed structures resembled large vesicles, which were the equivalent of 2-5 regular vesicle sizes. These results suggest that crayfish axon terminals recycle vast amounts of membrane in response to sustained 20-Hz stimulation and endocytosis appears to occur via multiple routes with the most common being through large vesicle intermediates. / Graduate