Marcy J. Bubar

Distribution of serotonin 5-HT2C receptors in the ventral tegmental area.

Serotonin 2C receptors (5-HT2CR) appear to exert tonic inhibitory influence over dopamine (DA) neurotransmission in the ventral tegmental area (VTA), the origin of the mesolimbic DA system, thought to be important in psychiatric disorders including addiction and schizophrenia. Current literature suggests that the inhibitory influence of 5-HT2CR on DA neurotransmission occurs via indirect activation of GABA inhibitory neurons, rather than via a direct action of 5-HT2CR on DA neurons. The present experiments were performed to establish the distribution of 5-HT2CR protein on DA and GABA neurons in the VTA of male rats via double-label immunofluorescence techniques. The 5-HT2CR protein was found to be co-localized with the GABA synthetic enzyme glutamic acid decarboxylase (GAD), confirming the presence of the 5-HT2CR on GABA neurons within the VTA. The 5-HT2CR immunoreactivity was also present in cells that contained immunoreactivity for tyrosine hydroxylase (TH), the DA synthetic enzyme, validating the localization of 5-HT2CR to DA neurons in the VTA. While the degree of 5-HT2CR+GAD co-localization was similar across the rostro-caudal levels of VTA subnuclei, 5-HT2CR+TH co-localization was highest in the middle relative to rostral and caudal levels of the VTA, particularly in the paranigral, parabrachial, and interfascicular subnuclei. The present results suggest that the inhibitory influence of the 5-HT2CR over DA neurotransmission in the VTA is a multifaceted and complex interplay of 5-HT2CR control of the output of both GABA and DA neurons within this region.

Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction.

Serotonin (5-HT) action via the 5-HT(2C) receptor (5-HT(2C)R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT(2C)R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT(2C)R. The expression of 5-HT(2C)R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT(2C)R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT(2C)R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT(2C)R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT(2C)R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT(2C)R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.

Prospects for serotonin 5-HT2R pharmacotherapy in psychostimulant abuse

The serotonin (5-HT) neurotransmitter system provides fundamental modulatory regulation of the limbic-corticostriatal circuitry known to be vital in the development of addiction as well as the aspects of addiction that hinder recovery and contribute to relapse. Thus, components of the 5-HT system may provide novel targets for the development of pharmacological treatments for psychostimulant dependence, which is associated with significant aberrations in dopamine (DA) neurotransmission. Two key modulators of DA signalling within the limbic-corticostriatal circuit are the 5-HT(2A) receptor (5-HT(2A)R) and the 5-HT(2C)R. These receptors are known to control the neurochemical and behavioural effects of psychostimulants, and in particular the in vivo effects of cocaine. Pre-clinical studies indicate that 5-HT(2A)R antagonists and/or 5-HT(2C)R agonists may effectively reduce craving and/or relapse, and likewise, enhance abstinence, while 5-HT(2C)R agonists may also effectively reduce cocaine intake in active cocaine users. At present, the progression of studies to probe the effectiveness of 5-HT(2A)R and 5-HT(2C)R ligands in the clinical setting is hindered by a lack of available, selective 5-HT(2A)R antagonists or 5-HT(2C)R agonists for use in human cocaine abusers. However, a number of selective 5-HT(2)R ligands currently under development, or in early clinical trials for psychiatric and/or neurological disorders, may soon be available for translational studies to explore their effectiveness in modulating drug use and dependence.

Serotonin 5-HT2A and 5-HT2C receptors as potential targets for modulation of psychostimulant use and dependence

The development of novel pharmacological agents for the treatment of psychostimulant use disorders is an important research imperative. One potential target system that has been largely overlooked is the serotonin (5-HT) neurotransmitter system. Preclinical studies indicate that 5-HT may be important in modulating the reinforcing properties of various drugs of abuse. While the potential sites of action of 5-HT within the brain are extensive, the natural starting point to examine the mechanisms by which 5-HT may be useful in treatment of psychostimulant use disorders is the interaction between 5-HT and dopamine (DA), a primary mediator of the rewarding effects of psychostimulants. Two key modulators of DA output are the serotonin (5-HT)2A receptor (5-HT2A R) and the 5-HT2C R. These receptors are known to control the neurochemical and behavioral effects of psychostimulants, and in particular, the in vivo effects of cocaine. Preclinical studies indicate that 5-HT2A R antagonists and/or 5-HT2C R agonists may effectively reduce craving and/or relapse, and likewise, enhance abstinence, while 5-HT2C R agonists may also effectively reduce cocaine intake in active cocaine users. At present, the progression of studies to probe the effectiveness of 5-HT2A R and 5-HT2C R ligands in the clinical setting is hindered by a lack of available selective 5-HT2A R antagonists or 5-HT2C R agonists for use in human cocaine abusers. However, a number of selective 5-HT2 R ligands currently under development, or in early clinical trials for psychiatric and/or neurological disorders, may soon be available for translational studies to explore their effectiveness in modulating drug use and dependence.

5-HT2C Receptors Localize to Dopamine and GABA Neurons in the Rat Mesoaccumbens Pathway

The serotonin 5-HT2C receptor (5-HT2CR) is localized to the limbic-corticostriatal circuit, which plays an integral role in mediating attention, motivation, cognition, and reward processes. The 5-HT2CR is linked to modulation of mesoaccumbens dopamine neurotransmission via an activation of γ-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA). However, we recently demonstrated the expression of the 5-HT2CR within dopamine VTA neurons suggesting the possibility of a direct influence of the 5-HT2CR upon mesoaccumbens dopamine output. Here, we employed double-label fluorescence immunochemistry with the synthetic enzymes for dopamine (tyrosine hydroxylase; TH) and GABA (glutamic acid decarboxylase isoform 67; GAD-67) and retrograde tract tracing with FluoroGold (FG) to uncover whether dopamine and GABA VTA neurons that possess 5-HT2CR innervate the nucleus accumbens (NAc). The highest numbers of FG-labeled cells were detected in the middle versus rostral and caudal levels of the VTA, and included a subset of TH- and GAD-67 immunoreactive cells, of which >50% also contained 5-HT2CR immunoreactivity. Thus, we demonstrate for the first time that the 5-HT2CR colocalizes in DA and GABA VTA neurons which project to the NAc, describe in detail the distribution of NAc-projecting GABA VTA neurons, and identify the colocalization of TH and GAD-67 in the same NAc-projecting VTA neurons. These data suggest that the 5-HT2CR may exert direct influence upon both dopamine and GABA VTA output to the NAc. Further, the indication that a proportion of NAc-projecting VTA neurons synthesize and potentially release both dopamine and GABA adds intriguing complexity to the framework of the VTA and its postulated neuroanatomical roles.

Selective serotonin 5-HT2C receptor activation suppresses the reinforcing efficacy of cocaine and sucrose but differentially affects the incentive-salience value of cocaine- vs. sucrose-associated cues

Serotonin (5-HT) controls affective and motivational aspects of palatable food and drug reward and the 5-HT(2C) receptor (5-HT(2C)R) has emerged as a key regulator in this regard. We have evaluated the efficacy of a selective 5-HT(2C)R agonist, WAY 163909, in cocaine and sucrose self-administration and reinstatement assays employing parallel experimental designs in free-fed rats. WAY 163909 dose-dependently reduced the reinforcing efficacy of cocaine (ID(50) = 1.19 mg/kg) and sucrose (ID(50) = 0.7 mg/kg) as well as reinstatement (ID(50) = 0.5 mg/kg) elicited by exposure to cocaine-associated contextual cues, but not sucrose-associated contextual cues. The ID(50) of WAY 163909 predicted to decrease the reinforcing efficacy of cocaine or sucrose as well as reinstatement upon exposure to cocaine-associated cues was ∼5-12-fold lower than that predicted to suppress horizontal ambulation (ID(50) = 5.89 mg/kg) and ∼2-5-fold lower than that predicted to suppress vertical activity (ID(50) = 2.3 mg/kg). Thus, selective stimulation of the 5-HT(2C)R decreases the reinforcing efficacy of cocaine and sucrose in freely-fed rats, but differentially alters the incentive-salience value of cocaine- vs. sucrose-associated cues at doses that do not impair locomotor activity. Future research is needed to tease apart the precise contribution of 5-HT(2C)R neurocircuitry in reward and motivation and the learning and memory processes that carry the encoding for associations between environmental cues and consumption of rewarding stimuli. A more complete preclinical evaluation of these questions will ultimately allow educated proof-of-concept trials to test the efficacy of selective 5-HT(2C)R agonists as adjunctive therapy in chronic health maladies including obesity, eating disorders and drug addiction.

Selective serotonin reuptake inhibitors enhance cocaine-induced locomotor activity and dopamine release in the nucleus accumbens.

The role for serotonin (5-HT) in mediating the behavioral effects of cocaine may be related in part to the ability of 5-HT to modulate the function of the dopamine (DA) mesoaccumbens pathways. In the present study, the ability of the selective serotonin reuptake inhibitors (SSRIs) fluoxetine (10 mg/kg, IP) and fluvoxamine (10 and 20 mg/kg, IP) to alter cocaine (10 mg/kg, IP)-induced hyperactivity and DA release in the nucleus accumbens (NAc) was analyzed in male Sprague-Dawley rats. Systemic administration of either fluoxetine or fluvoxamine enhanced cocaine-induced locomotor activity in a dose-dependent manner; fluoxetine (10 mg/kg, IP) also enhanced cocaine (10 mg/kg, IP)-induced DA efflux in the NAc. To test the hypothesis that the NAc serves as the locus of action underlying these effects following systemic cocaine administration, fluoxetine (1 and 3 micro g/0.2 micro l/side) or fluvoxamine (1 and 3 micro g/0.2 micro l/side) was microinfused into the NAc shell prior to systemic administration of cocaine (10 mg/kg, IP). Intra-NAc shell infusion of 3 micro g of fluoxetine or fluvoxamine enhanced cocaine-induced hyperactivity, while infusion of fluoxetine (1 micro M) through the microdialysis probe implanted into the NAc shell enhanced cocaine (10 mg/kg, IP)-induced DA efflux in the NAc. Thus, the ability of systemic injection of SSRIs to enhance cocaine-evoked hyperactivity and DA efflux in the NAc is mediated in part by local actions of the SSRIs in the NAc.

Contribution of serotonin (5-HT) 5-HT2 receptor subtypes to the discriminative stimulus effects of cocaine in rats

RationaleThe serotonin (5-hydroxytryptamine; 5-HT) 5-HT2 receptor (5-HT2R) family is an important regulator of the behavioral responsiveness to cocaine.ObjectiveThe present study is an analysis of the role of the 5-HT2R subtypes (5-HT2AR, 5-HT2BR, and 5-HT2CR) in the discriminative stimulus effects of cocaine.MethodsMale Wistar rats were trained to discriminate cocaine (10xa0mg/kg) from saline in a two-lever, water-reinforced FR 20 task, and we investigated the ability of the 5-HT2AR antagonist 1(Z)-[2-(dimethylamino)ethoxyimino]-1(2-fluorophenyl)-3-(4-hydroxyphenyl)-2(E)-propene (SR 46349B), the 5-HT2BR antagonist N-(1-methyl-5-indolyl)-N′-(3-methyl-5-isothiazolyl) urea (SB 204741), and the 5-HT2CR antagonist [(+)-cisxa0-4,5,7a,8,9,10,11,11a-octahydro-7H-10-methylindolo(1,7-bC)(2,6)naphthyridine (SDZ SER-082) to substitute for or to modulate the stimulus effects of cocaine.ResultsPretreatment with SR 46349B (0.5–1xa0mg/kg) resulted in a rightward shift of the cocaine dose–response curve, while SDZ SER-082 (1xa0mg/kg) shifted the dose–response for cocaine to the left; SB 204741 (1–3xa0mg/kg) was inactive.ConclusionsOur pharmacological analyses of selective antagonists of 5-HT2AR, 5-HT2BR, and 5-HT2CR indicate oppositional influence of 5-HT2AR and 5-HT2CR on the stimulus effects of cocaine and exclude a role for the 5-HT2BR. These data suggest that 5-HT2AR and 5-HT2CR may be important in modulating the subjective effects of cocaine in humans.

Effects of dopamine D1- or D2-like receptor antagonists on the hypermotive and discriminative stimulus effects of (+)-MDMA.

RationaleBoth dopamine (DA) and serotonin (5-HT) release are evoked by (+)-MDMA; however, little is known of the contribution of DA D1- and D2-like receptors (D1R and D2R, respectively) in the behavioral effects of (+)-MDMA.ObjectivesTo test the hypothesis that a D1R or D2R antagonist would attenuate the hypermotive or discriminative stimulus effects of (+)-MDMA.MethodsMale Sprague-Dawley rats (n=164) were pretreated with the D1R antagonist SCHxa023390 (3.125–50xa0μg/kg, SC) or the D2R antagonist eticlopride (12.5–50xa0μg/kg, SC) prior to treatment with (+)-MDMA (3xa0mg/kg, SC) and locomotor activity was recorded using photobeam monitors. Twelve additional rats trained to discriminate (+)-MDMA (1xa0mg/kg, IP) from saline in a two-lever water-reinforced FR20 task were administered SCHxa023390 (6.25xa0μg/kg, IP) or eticlopride (12.5xa0μg/kg, IP) prior to (+)-MDMA (0.375–1.0xa0mg/kg, IP). Rats were then placed in the drug discrimination chambers and the percent (+)-MDMA appropriate responding and response rate were measured.ResultsBoth SCHxa023390 and eticlopride blocked (+)-MDMA-evoked hyperactivity in a dose-related manner; the highest doses of the antagonists also effectively suppressed basal locomotor activity. In rats trained to discriminate (+)-MDMA from saline, SCHxa023390 (6.25xa0μg/kg), but not eticlopride (12.5xa0μg/kg), blocked the stimulus effects of (+)-MDMA without altering response rate.ConclusionThese data indicate that DA released indirectly by (+)-MDMA administration results in stimulation of D1R and D2R to enhance locomotor activity. Furthermore, the D1R appears to play a more prominent role than the D2R in the discriminative stimulus properties of (+)-MDMA.

Synergism between a serotonin 5-HT2A receptor (5-HT2AR) antagonist and 5-HT2CR agonist suggests new pharmacotherapeutics for cocaine addiction.

Relapse to cocaine dependence, even after extended abstinence, involves a number of liability factors including impulsivity (predisposition toward rapid, unplanned reactions to stimuli without regard to negative consequences) and cue reactivity (sensitivity to cues associated with cocaine-taking which can promote cocaine-seeking). These factors have been mechanistically linked to serotonin (5-hydroxytryptamine, 5-HT) signaling through the 5-HT(2A) receptor (5-HT(2A)R) and 5-HT(2C)R; either a selective 5-HT(2A)R antagonist or a 5-HT(2C)R agonist suppresses impulsivity and cocaine-seeking in preclinical models. We conducted proof-of-concept analyses to evaluate whether a combination of 5-HT(2A)R antagonist plus 5-HT(2C)R agonist would have synergistic effects over these liability factors for relapse as measured in a 1-choice serial reaction time task and cocaine self-administration/reinstatement assay. Combined administration of a dose of the selective 5-HT(2A)R antagonist M100907 plus the 5-HT(2C)R agonist WAY163909, each ineffective alone, synergistically suppressed cocaine-induced hyperactivity, inherent and cocaine-evoked impulsive action, as well as cue- and cocaine-primed reinstatement of cocaine-seeking behavior. The identification of synergism between a 5-HT(2A)R antagonist plus a 5-HT(2C)R agonist to attenuate these factors important in relapse indicates the promise of a bifunctional ligand as an anti-addiction pharmacotherapeutic, setting the stage to develop new ligands with improved efficacy, potency, selectivity, and in vivo profiles over the individual molecules.