Dorsal raphe 5-HT2C receptor and GABA networks regulate anxiety produced by cocaine withdrawal
Introduction
Severe anxiety provides part of the negative reinforcement associated with cocaine dependence and contributes to relapse and maintenance of cocaine abuse (Markou and Koob, 1992). The serotonin system regulates anxiety, and it is possible that long-term alterations in serotonin activity elicited by cocaine exposure contribute to anxiety during withdrawal (Darmani et al., 1997, Parsons et al., 1995). The 5-HT2C receptor (5-HT2CR) regulates the effects of cocaine, as activation of the 5-HT2CR reduces cocaine-induced increases in serotonin and dopamine neurotransmission (Di Matteo et al., 2000, Navailles et al., 2007). Separately, a role for the 5-HT2CR in the orchestration of anxiety-like behaviors has been identified. Pharmacological blockade of 5-HT2CRs prevents anxiety expression (Christianson et al., 2010). These studies implicate a direct role for 5-HT2CRs in regulating anxiety and cocaine effects, suggesting a putative target for studying the link between serotonin regulation and anxiety produced by cocaine withdrawal.
The inhibition of dopamine and serotonin neurotransmission by 5-HT2CRs may arise via an indirect modulation of gamma-aminobutyric acid (GABA) neurotransmission. 5-HT2CR located on GABA interneurons (Serrats et al., 2005) constitute a negative feedback circuit that mediates 5-HT2CR-induced suppression of dorsal raphe (DR) serotonin (Boothman et al., 2006, Quérée et al., 2009), which regulates anxiety neurocircuitry (Spoida et al., 2014). The DR, the primary source of serotonin in the forebrain, is integral in regulating cocaine withdrawal-induced anxiety (Ettenberg et al., 2011). Dysfunction of the negative feedback function of the 5-HT2CR and GABA activity could interfere with DR activity, and disrupt serotonin signaling in contribution to cocaine withdrawal-induced anxiety.
In the present study, anxiety during cocaine withdrawal was assessed in the context of DR 5-HT2CR and GABA function. DR serotonin neurons are heterogeneous, existing in three distinct regional subpopulations with varying function and projections to forebrain regions (Calizo et al., 2011). The serotonin-populated dorsomedial and ventromedial DR project to both subcortical and cortical structures, and in contrast, the lateral wing subregion, densely populated by GABA neurons, innervates only subcortical structures (Azmitia and Segal, 1978, Kirifides et al., 2001, Muzerelle et al., 2014). Investigation of the differences in cell activity and characteristics in each subregion during cocaine withdrawal provides a mechanistic description of the DR serotonin neurocircuitry and its influence on withdrawal-induced anxiety.
The current study used a binge cocaine model to mimic the cocaine use pattern often seen in human cocaine addicts, while exercising control over total drug exposure and producing consistent withdrawal behavior. Binge-pattern cocaine administration produces similar neuroadaptations in rodents and humans, and results in withdrawal-induced anxiety (Basso et al., 1999, Gawin and Kleber, 1986, Mutschler and Miczek, 1998, Perrine et al., 2008). To our knowledge, we are the first to characterize the physiology of DR serotonin neuron subpopulations in the regulation of anxiety produced by cocaine withdrawal, and to identify a role for 5-HT2CR and GABA networks in mediating cocaine withdrawal-induced anxiety. Through targeting acute and protracted stages of withdrawal, this research identifies a functional mechanism for the production of cocaine withdrawal-induced anxiety, and suggests novel pharmacological targets for prevention of relapse.
Section snippets
Animals
Adult male C57Bl/6 mice (22–24 g at the start of the experiment) were obtained from Charles River, Inc. (Wilmington, MA) and housed 4 per cage. Mice were maintained on a 12 h light/dark cycle and provided food and water ad libitum. All testing was accomplished during the light phase. Animal use procedures were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Temple University.
Drugs
Cocaine
Acute withdrawal from repeated cocaine increased anxiety-like behaviors
Mice were tested on the elevated plus maze to assess anxiety-like behavior during active cocaine exposure (30 min following the last injection) or at 25 h or 7 days of withdrawal. The data in Fig. 1A represent the mean time spent on the open arms as a percentage of the total time on the elevated plus maze. Two-way ANOVA revealed a significant interaction between treatment and withdrawal time (interaction, F(2,84) = 4.518, p = 0.0137; treatment, F(1,84) = 11.87, p = 0.0009; withdrawal time, F
Discussion
In the current study, elevated plus maze testing identified an anxiogenic phenotype in cocaine-injected mice at 24 h of cocaine withdrawal, an effect that was absent at 30 min or 7 days after the last cocaine injection. Electrophysiology studies showed that DR serotonin neurons from anxious, cocaine-treated mice in acute 24 h withdrawal demonstrated heightened sIPSC and mIPSC frequencies in the dmDR and vmDR. This effect was blocked in DR serotonin cells upon bath application of the 5-HT2CR
Acknowledgments
This work was supported in part by NIDA/NIH: R01 DA018326 (EMU) and P30 DA013429 (EMU/LGK).
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