Regulation of Cocaine-induced Behaviors and Anxiety Produced by Cocaine Withdrawal through the Serotonin(2C) Receptor

Abstract

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.