From obesity to substance abuse: therapeutic opportunities for 5-HT2C receptor agonists  Guy A. Higgins, Edward M. Sellers, Paul J. Fletcher  Trends in Pharmacological Sciences  Volume 34, Issue 10, Pages 560-570 (October 2013) DOI: 10.1016/j.tips.2013.08.001 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Three brain regions where 5-HT2C receptor agonists may modulate behaviors relating to feeding and drug taking. (A) 5-HT2C receptors are expressed in ventral tegmental area (VTA) [103]. The upper panel shows 5-HT2C immunoreactivity (IR, red color) colocalized with GAD IR or TH IR (green color) in subregions of the VTA (from [103] with permission). These receptors modulate activity of ascending dopaminergic (DA) pathways to the nucleus accumbens (N.Acc) to alter motivation and ‘drive’ for both primary and secondary reinforcers. Support for this pathway comes from findings that infusion of 5-HT2C receptor agonists into the VTA reduces cocaine induced locomotion, cocaine self-administration and cocaine induced DA overflow in N.Acc [104,105]. Local intra-VTA infusion of Ro60-0175 also blunts stress induced increases in medial PFC (mPFC) DA overflow [106]. The lower panel shows a schema illustrating that activation of 5-HT2C receptors localized on inhibitory GABAergic [glutamic acid decarboxylase (GAD) +ve] interneurons increases GABA release onto GABA-A receptors on DA VTA neurons with consequent reduced firing and terminal DA release. 5-HT2C receptors directly located on DA cell bodies [tyrosine hydroxylase (TH) +ve] may also influence VTA cell firing via 5-HT inputs from, for example, the dorsal or median raphe nucleus (DRN/MRN). Also shown is how the nicotine agonist varenicline may similarly drive inhibitory GABA tone via α4β2 interaction. Stimulation of α4β2 channels within the VTA by nicotine is thought to contribute to abuse property. As a partial α4β2 agonist, varenicline may dampen this property. (B) 5-HT2C receptors are expressed in mPFC [107]. Interactions between 5-HT2C agonists and GABAergic and/or glutamatergic systems within the PFC may regulate behaviors such as impulsivity and reinstatement of stimulus seeking behavior. The upper panel shows 5-HT2C IR (red color) colocalized with GAD IR (green color) in subregions of the PFC (e.g., prelimbic); (from [107] with permission). Support for this pathway comes from observations that infusion of 5-HT2C receptor agonists into mPFC reduces reinstatement of cocaine-seeking [108], and that altered mPFC activity influences impulsive behavior [109]. The lower panel shows a proposed schema illustrating how 5-HT2C receptors localized on GABA interneurons (e.g., GAD +ve) localized within cortical regions may inhibit the tone of descending glutamatergic excitatory inputs to structures such as N.Acc and VTA (adapted from [107]). Also, 5-HT2C receptors may exert inhibitory control over DRN cell firing (see [110]). (C) 5-HT2C receptors are expressed in the hypothalamus. Within the arcuate nucleus (ARC) of the hypothalamus, 5-HT2C receptor-mediated interactions with melanocortin are important for the regulatory control of food intake. The upper panel shows 5-HT2C mRNA (black dots) colocalized with αMSH-IR neurons (brown color) in arcuate nucleus of the hypothalamus; panel from [30] with permission. Support for this pathway comes from findings such as microinjection of 5-HT2 agonists such as TFMPP into the hypothalamus reduce feeding [111], and selective re-expression of 5-HT2C receptors in proopiomelanocortin (POMC) neurons (albeit not limited to the ARC) can restore anorectic effects of dexfenfluramine rendered absent in global 5-HT2C receptor-null mice [112]. The lower panel depicts how 5-HT may activate 5-HT2C receptors located on POMC neurons in the ARC to release αMSH which activates MC4R receptors in PVN to promote satiety. Also, 5-HT1B receptors located on AgRP/NPY neurons may regulate PVN neurons. 5-HT input is likely to originate from the DRN (adapted from [9]). Trends in Pharmacological Sciences 2013 34, 560-570DOI: (10.1016/j.tips.2013.08.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Summary of effect of the 5-HT2C receptor agonist, Ro60-0175, against various behaviors under the control of acute or chronic nicotine administration. (A) The intravenous self-administration of nicotine (0.03mg/kg/infusion) under FR5 or progressive ratio (PR) schedules of reinforcement is reduced by acute Ro60-0175 (0.3–1mg/kg), an effect that is reversed by SB-242084 pretreatment. Adapted from Fletcher et al. (2012) [58], with permission. (B) The reinstatement of nicotine-seeking behavior induced either by a nicotine priming injection (right), or light/tone cue (left) is reduced by acute Ro60-0175 (0.3–1mg/kg), an effect that is reversed by SB-242084 pretreatment. Adapted from Fletcher et al. (2012) [58], with permission. (C) In rats implanted with osmotic minipumps primed to release nicotine at either 6 or 9mg/kg/day, the acute administration of mecamylamine (1mg/kg subcutaneous, s.c.) after 9 days elicits behaviors likely reflecting somatic withdrawal signs. Ro60-0175 (1mg/kg s.c.) failed to affect the expression of these signs [59]. (D) In a test of impulsive action on the 5-CSRT test increasing the interval (ITI) between trials from 5s to 9s increased premature responding. Ro60-0175 (0.3mg/kg) reduced this behavior without detrimentally affecting other aspects of task performance. Adapted from Fletcher et al. (2007) [62], with permission. (E) In rats trained to discriminate nicotine (0.3mg/kg) from saline, Ro60-0175 (0.6mg/kg) reduced the cueing effect of nicotine. Adapted from Higgins et al. (2013) [32], with permission. (F) Time course of the effect of acute nicotine (1mg/kg intraperitoneally, i.p.) on extracellular dopamine levels in the nucleus accumbens (N.Acc) measured by microdialysis. Rats were treated for 10 days with nicotine (1mg/kg i.p.) prior to the microdialysis experiment. Rats were treated with either vehicle or Ro60-0175 (1mg/kg i.p.) 20min before nicotine (1mg/kg i.p.) or vehicle at 0min. Nicotine increased extracellular dopamine release in the N.Acc. This effect was significantly attenuated by Ro60-0175. Adapted from Di Matteo et al. (2004) [51], with permission. (G) Effect of nicotine (intravenous, i.v.) and Ro60-0175 (0.1mg/kg i.v) on the firing pattern on VTA dopamine neurons of rats treated for 10 days with nicotine (1mg/kg i.p.). The data represent the mean+SEM difference between the percentage of spikes occurring in bursts during baseline period compared to post drug treatment. The data show that nicotine increased the burst firing of VTA dopamine neurons, and this effect was blocked by Ro60-0175. Data adapted from Pierucci et al. (2004) [52], with permission. Abbreviations: Ro, Ro60-0175; Nic, nicotine; Veh, vehicle. Trends in Pharmacological Sciences 2013 34, 560-570DOI: (10.1016/j.tips.2013.08.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure I The bipolar visual analog scale (VAS) is one of the most validated and predictive scales for abuse post-marketing. Least mean square differences (+/– 95% CI) from placebo on the bipolar VAS anchored at zero with positive changes reflecting liking and negative changes reflecting disliking. *, P<0.05 compared to placebo. Redrawn from Table 1 in Schram et al. [74], with permission. Trends in Pharmacological Sciences 2013 34, 560-570DOI: (10.1016/j.tips.2013.08.001) Copyright © 2013 Elsevier Ltd Terms and Conditions