Attenuation of cue-induced heroin-seeking by the atypical antidepressant mirtazapine.

High-speed chronoamperometry with monoamine-selective carbon fiber electrodes was used in rats to monitor, during 5-6 consecutive daily sessions, changes in DA-dependent electrochemical signal in the nucleus accumbens (NAcc) during intravenous heroin (0.1 mg/kg) self-administration (SA) behavior and passive repeated drug injections performed with a temporal scheme similar to that in the SA experiment. In trained animals, biphasic signal fluctuations time-locked to the individual lever-presses were found to accompany all but the first daily SAs. The signal gradually increased by 30-40 nM for the 10 minutes preceding the SA, reached a peak at the moment of lever-press and decreased abruptly by approximately 40 nM for 3-4 min after heroin SA. The cycle then repeated, reaching a new peak at the moment of the next lever-press. Rapid bi-directional fluctuations in signal associated with individual heroin SAs were superimposed on substantial tonic increase in signal baseline (400-500 nM). This increase quickly developed after presentation of heroin-related light cue and the first SA, was relatively stable during all subsequent SAs and decreased towards the baseline after the last SA of a session. Changes in signal baseline induced by repeated heroin SAs depended strongly upon the signal's basal level (r = -0.787); that signal preferentially increased when its basal values were low (0-300 nM), and decreased when signal was tonically elevated (> 600 nM). Repeated passive heroin injections also induced biphasic signal fluctuations and a similar tonic increase in signal baseline. Although a transient signal decrease (25 nM for 2-4 minutes) followed by a prolonged signal increase occurred after each but not the first passive injection, the gradual pre-injection signal acceleration was absent. Although DOPAC, a principal DA metabolite, may significantly contribute to the tonic increase in electrochemical signal seen during SA session, the changes in extracellular DA may be the main contributor to both the rapid signal increases preceding drug-taking and the transient signal decreases following heroin SA. If so, the present findings suggest that activation of mesolimbic DA cells and increase in DA transmission may be involved in the mediation of motivational and/or activational components of drug-seeking and drug-taking behavior. An acute termination of previous drug- and behavior-associated DA activation with a transient inhibition of DA release, immediately following heroin SA may correlate with the drug's rewarding action, representing a part of a mechanism regulating drug-taking behavior.

Clinical and preclinical studies indicate that adaptations in corticostriatal neurotransmission significantly contribute to heroin relapse vulnerability. In animal models, heroin self-administration and extinction produce cellular adaptations in both neurons and astrocytes within the nucleus accumbens (NA) core that are required for cue-induced heroin seeking. Specifically, decreased glutamate clearance and reduced association of perisynaptic astrocytic processes with NAcore synapses allow glutamate release from prelimbic (PrL) cortical terminals to engage synaptic and structural plasticity in NAcore medium spiny neurons. Normalizing astrocyte glutamate homeostasis with drugs like the antioxidant N-acetylcysteine (NAC) prevents cue-induced heroin seeking. Surprisingly, little is known about heroin-induced alterations in astrocytes or pyramidal neurons projecting to the NAcore in the PrL cortex (PrL-NAcore). Here, we observe functional adaptations in the PrL cortical astrocyte following heroin self-administration (SA) and extinction as measured by the electrophysiologically evoked plasmalemmal glutamate transporter 1 (GLT-1)-dependent current. We likewise observed the increased complexity of the glial fibrillary acidic protein (GFAP) cytoskeletal arbor and increased association of the astrocytic plasma membrane with synaptic markers following heroin SA and extinction training in the PrL cortex. Repeated treatment with NAC during extinction reversed both the enhanced astrocytic complexity and synaptic association. In PrL-NAcore neurons, heroin SA and extinction decreased the apical tuft dendritic spine density and enlarged dendritic spine head diameter in male Sprague-Dawley rats. Repeated NAC treatment during extinction prevented decreases in spine density but not dendritic spine head expansion. Moreover, heroin SA and extinction increased the co-registry of the GluA1 subunit of AMPA receptors in both the dendrite shaft and spine heads of PrL-NAcore neurons. Interestingly, the accumulation of GluA1 immunoreactivity in spine heads was further potentiated by NAC treatment during extinction. Finally, we show that the NAC treatment and elimination of thrombospondin 2 (TSP-2) block cue-induced heroin relapse. Taken together, our data reveal circuit-level adaptations in cortical dendritic spine morphology potentially linked to heroin-induced alterations in astrocyte complexity and association at the synapses. Additionally, these data demonstrate that NAC reverses PrL cortical heroin SA-and-extinction-induced adaptations in both astrocytes and corticostriatal neurons.

A series of simple O-acylated derivatives of the naturally occurring clavine alkaloids lysergol and dihydrolysergol-I were synthesized and tested in-vitro for their ability to interact with 5-HT2A receptors in rat tail artery, 5-HT2C receptors in piglet choroid plexus, 5-HT1B receptors in guinea-pig iliac artery and alpha1-adrenergic receptors in rat aorta. In contrast to the classical ergoline 5-HT2A receptor antagonists methysergide and LY53857, the compounds produced competitive antagonism of the 5-HT response in rat tail artery. Affinities of ergolines 3-14 were higher (pA2 values of 7.33-8.40) than those of the parent alcohols lysergol (1) and dihydrolysergol-I (2), respectively. The introduction of an isopropyl substituent at the N(1) position of the compounds failed to enhance 5-HT2A receptor affinity. Compounds 3-14 exhibited lower affinities for alpha1-adrenergic receptors than for 5-HT2A receptors. In particular, those lysergol derivatives that had an isopropyl substituent at the N(1) position were highly specific 5-HT2A receptor antagonists (ratio 5-HT2A/alpha1 = 302-3548). Selected derivatives of lysergol (3-5, 9-11) which were assayed for radioligand binding at 5-HT2C receptors in piglet choroid plexus had affinities that were similar to those found in rat tail artery. Additionally, lysergol and its N(1)-unsubstituted derivatives were found to be partial agonists (alpha of 0.2-0.4) for 5-HT2C receptor-mediated inositol phosphate accumulation in piglet choroid plexus. On the other hand, analogues with an isopropyl substituent at N(1) showed no measurable agonist activity. The observation that N(1)-unsubstituted derivatives of lysergol possessed agonist properties at 5-HT2C receptors whereas their agonist activity at 5-HT2A receptors was marginal (alpha of 0.05 for compound 3 at 1 microM) or not measurable, suggests that these compounds have different abilities to cause conformational change at the two receptor types. Selected derivatives of lysergol (3-5, 9-11) which were examined as ligands for 5-HT1B receptors in guinea-pig iliac artery caused insurmountable blockade of the contractile effect of 5-HT. N(1)-isopropyl derivatives had 30-50-fold lower affinities for 5-HT1B receptors of this tissue than their N(1)-unsubstituted analogues. It is concluded that O-acylated derivatives of the clavine alkaloids lysergol and dihydrolysergol-I mimic therapeutically relevant ergolines due to the complexity of their pharmacological profile as partial agonists and antagonists at 5-HT2A, 5-HT2C and 5-HT1B receptors, and at alpha1-adrenergic receptors.

Lorcaserin is a serotonin (5-HT)2C receptor-preferring agonist approved by the US Food and Drug Administration to treat obesity. Lorcaserin decreases cocaine self-administration in rats and monkeys. Although this effect is partially inhibited by a 5-HT2C receptor antagonist (SB242084), lorcaserin also has effects at 5-HT2A and 5-HT1A receptors, and the relative contribution of these receptors to its anti-cocaine effects has not been investigated. The goals of this study were to determine 1) the potency and effectiveness of lorcaserin to decrease self-administration of cocaine and 3,4-methylenedioxypyrovalerone (MDPV), a common “bath salts” constituent; and 2) the receptor(s) mediating the effects of lorcaserin on cocaine and MDPV self-administration. Male Sprague-Dawley rats (n = 6) were trained to self-administer MDPV under a progressive ratio schedule of reinforcement and maintained under this schedule with daily access to 0.32 mg/kg per infusion of cocaine or 0.032 mg/kg per infusion of MDPV. Dose-response curves for the effects of lorcaserin on cocaine and MDPV self-administration were generated by administering lorcaserin (0.1–5.6 mg/kg) 25 minutes before the start of the session. To assess the effects of 5-HT2C (SB242084, 0.1 mg/kg), 5-HT2A (MDL100907, 0.1 mg/kg), and 5-HT1A (WAY100635, 0.178 mg/kg) receptor antagonists, they were administered 15 minutes before lorcaserin. Lorcaserin decreased cocaine and MDPV self-administration with equal potency. Antagonism of 5-HT2C (but not 5-HT1A or 5-HT2A) receptors blocked the effects of lorcaserin on cocaine and MDPV self-administration. Taken together, these data provide additional support for further development of 5-HT2C receptor agonists, such as lorcaserin, for the treatment of stimulant abuse.

Evidence from studies with phenylisopropylamine hallucinogens indicates that the 5HT2A receptor is the likely target for the initiation of events leading to hallucinogenic activity associated with LSD and related drugs. Recently, lisuride (a purported non-hallucinogenic congener of LSD) was reported to be a potent antagonist at the 5HT2C receptor and an agonist at the 5HT2A receptor. LSD exhibited agonist activity at both receptors. These data were interpreted as indicating that the 5HT2C receptor might be the initiating site of action for hallucinogens. To test this hypothesis, recombinant cells expressing 5HT2A and 5HT2C receptors were used to determine the actions of LSD and lisuride. LSD and lisuride were potent partial agonists at 5HT2A receptors with EC50 values of 7.2 nM and 17 nM, respectively. Also, LSD and lisuride were partial agonists at 5HT2C receptors with EC50 values of 27 nM and 94 nM, respectively. We conclude that lisuride and LSD have similar actions at 5HT2A and 5HT2C receptors in recombinant cells. As agonist activity at brain 5HT2A receptors has been associated with hallucinogenic activity, these results indicate that lisuride may possess hallucinogenic activity, although the psychopharmacological effects of lisuride appear to be different from the hallucinogenic effects of LSD.

Serotonin-2C and -2a receptor co-expression on cells in the rat medial prefrontal cortex

1. We have used previously characterized clones of the human neuroblastoma cell line, SH-SY5Y. constitutively expressing either the human 5-HT2A or 5-HT2C receptor to compare their desensitization profiles after exposure to 5-HT. 2. 5-HT stimulated [3H]-inositol phosphate ([3H]-IPx) production at both the 5-HT2C (pEC50=8.03+/-0.15) and 5-HT2A receptors (pEC50=7.15+/-0.08), with maximal responses occurring after exposure to 1 microM and 10 microM 5-HT, respectively. 3. Exposure of cells to maximally effective concentrations of 5-HT caused time- and concentration-dependent desensitization of [3H]-IPx formation. The 5-HT2A response desensitized slower (t1/2 = 110 min) and with lower sensitivity than that of the 5-HT2C receptor (t1/2 = 12.5 min). In each case, desensitization was blocked by co-administration of a specific receptor antagonist. Following exposure to 10 microM 5-HT for 2 h, both receptors exhibited extensive desensitization, with subsequent responses to 5-HT reduced by more than 80%. 4. 5-HT stimulated Ins(1,4,5)P3 production with a potency similar to that for [3H]-IPx production at each receptor. In both cases Ins(1,4,5)P3 levels peaked rapidly then returned to basal level within a short time. This peak consistently occurred earlier for the 5-HT2C receptor (5 s) than for the 5-HT2A receptor (20 s). 5. Prior exposure of SH-SY5Y/5-HT2C cells to 5-HT (1 microM/15 min) caused a significant decrease in the 5-HT-stimulated peak in Ins(1,4,5)P3 levels whereas no such change occurred in SH-SY5Y/5-HT2A cells following exposure to 10 microM 5-HT for 15 min. 6 These results indicate that the human 5-HT2A and 5-HT2C receptors both exhibit desensitization at the level of inositol phosphate formation when expressed in the same cellular environment, with the 5-HT2C receptor being more sensitive to 5-HT-mediated desensitization than the 5-HT2A receptor.

Two complimentary techniques were used to estimate occupancy of pimavanserin (a selective 5-HT2A/2C inverse agonist) to 5-HT2A and 5-HT2C receptors in non-human primate brains. One employed the 5-HT2A/2C selective radioligand [11C]CIMBI-36 combined with quantification of binding potentials in brain regions known to be enriched in 5-HT2A (cortex) or 5-HT2C (choroid plexus) receptors to estimate occupancy. Pimavanserin was 6–10 fold more potent displacing [11C]CIMBI-36 from cortex (ED50 = 0.007 mg/kg; EC50 = 0.6 ng/ml) than from choroid plexus (ED50 =0.046 mg/kg; EC50 = 6.0 ng/ml). The assignment of [11C]CIMBI-36 binding to 5-HT2A and 5-HT2C receptors by anatomical brain structure was confirmed using the 5-HT2A selective inverse agonist MDL 100,907 and the 5-HT2C selective antagonist SB 242584 to displace [11C]CIMBI-36. The second technique employed a novel, 5-HT2C selective tracer called [11C]AC1332. [11C]AC1332 bound robustly to choroid plexus, moderately to hippocampus, and minimally to cortex. Pimavanserin displaced [11C]AC1332 with similar potency (ED50 = 0.062 mg/kg; EC50 = 2.5 ng/ml) as its potency displacing [11C]CIMBI-36 binding from choroid plexus. These results demonstrate the feasibility of simultaneously estimating drug occupancy of 5-HT2A and 5-HT2C receptors in vivo, and the utility of a novel 5-HT2C receptor selective tracer ligand.

G protein-coupled receptors (GPCRs) not only interact with heterotrimeric G proteins but also with accessory proteins, called GPCR-interacting proteins (GIPs). GIPs are implicated in GPCR targeting to specific cellular compartments, in their assembling into large functional complexes called “receptosomes,” in their trafficking to and from the plasma membrane, as well as in the fine-tuning of their signaling properties. Here, we describe “receptosomes“ associated with the C-terminal tails of 5-hydroxytryptamines 5-HT2A, 5-HT2C, as well as 5-HT4a and 5-HT4e receptors. The three last residues of these receptor C-termini are canonical PDZ ligands interacting with type I PDZ domain-containing proteins (5-HT2A, 5-HT2C, 5-HT4A tails) and type II (5-HT4e). The entire C-terminal tails fused to glutathione-S-transferase or synthetic peptides encompassing the last 14 C-terminal residues of the receptors were used as baits to fish out GIPs from mouse brain. Controls were made with mutant bait (mutated in the PDZ ligand). Proteins, which were specifically retained on native PDZ ligand-containing peptides, were separated on two-dimensional gels and identified by MALDI-TOF mass spectrometry or immunoblotting. Ten and seven PDZ domain-containing proteins were found to bind to the 5-HT2C and 5-HT2A receptors, respectively. The sequences of the C-terminal PDZ ligands of 5-HT2C and 5-HT2A receptors are very similar (SSV and SCV, respectively). If some of the PDZ domain-containing proteins associated with these receptors were identical (ARIP-1/MAGI-2, SAP97, PSD-95, Dlgh3/MPP3), others were clearly different. The 5-HT2C but not the 5-HT2A receptor interacted with the SAP102 and Veli3/CASK/Mint1 ternary complex, whereas the 5-HT2A but not the 5-HT2C receptors interacted with CIPP. MUPP1, which was found to interact with the 5-HT2C receptor in a two-hybrid screen, was also fished out by the 5-HT2C and 5-HT2A C-termini. A few other non-PDZ domain-containing proteins were found in these “receptosomes.” Electron microscopy (EM) studies of 5-HT2A and 5-HT2C receptors and some of their interacting proteins led to the proposition of a presynaptic and postsynaptic localization of 5-HT2C receptors and a preferential postsynaptic localization of 5-HT2A receptors. In a similar manner, we identified 10 and 3 proteins that interacted specifically with the 5-HT4a and 5-HT4e receptor splice variants, respectively. Most of them are PDZ proteins. Among them, NHERF recruited 5-HT4a receptors in microvilli, where they localized with activated ezrin, consistent with a role of 5-HT4a receptors in cytoskeleton remodeling. The same variant interacted with both the constitutive and the inducible (upon metham-phetamine treatment) forms of SNX27 (SNX27a and SNX27b, respectively). SNX27a redirected part of the 5-HT4a receptors to early endosomes.Key Words:GPCRs5-HT5-HT receptorsPDZ proteinsproteomics.

Deramciclane (EGIS-3886) is a novel anxiolytic agent that binds with high affinity to 5-HT2A/2C receptors. The interactions of deramciclane with the serotonin 5-HT2C receptor were characterized further using receptor phosphoinositide hydrolysis assays and receptor autoradiography. Deramciclane antagonized 5-HT2C receptor mediated 5-HT-stimulated phosphoinositide hydrolysis with an IC50 value of 168 nM. Deramciclane also decreased basal phosphoinositide hydrolysis by up to 33% (EC50 = 93 nM) in a physiological system in the choroid plexus, suggesting that deramciclane possesses inverse agonist properties at this receptor. Administration of single doses of 0.5 mg/kg and 10 mg/kg resulted in a maximal 5-HT2C receptor occupancy of up to 45% and 79%, respectively, in the choroid plexus. Chronic (14 days) treatment with 0.5 mg/kg or 10 mg/kg deramciclane did not alter [125I]DOI (agonist) or [3H]mesulergine (antagonist) binding to 5-HT2C receptors in the choroid plexus compared to saline-treated controls, as determined by quantitative receptor autoradiography. In comparison, the effects of deramciclane on 5-HT2A binding characteristics and receptor occupancy were also studied. Deramciclane treatment resulted in 5-HT2A receptor occupancy of up to 78%, but no significant effect of chronic treatment on 5-HT2A receptor agonist binding levels was found. In conclusion, these data indicate that deramciclane is a 5-HT2C receptor inverse agonist and occupies 5-HT2C receptors during treatment, and that chronic treatment with deramciclane does not lead to 5-HT2C receptor down-regulation.

Many modern models of receptor-G protein function assume that there is a direct relationship between high-affinity agonist binding and efficacy. The validity of this assumption has been recently questioned for the serotonin 5-HT2A receptor. We examined the intrinsic activities of various ligands in activating phosphoinositide hydrolysis and measured their respective binding affinities to the high- and low-affinity states of the 5-HT2C (VNV isoform) and 5-HT(2A) receptors. Ligand binding affinities for the high-affinity state of the receptors were determined using 1-(4-[125I]iodo-2,5-dimethoxyphenyl)2-aminopropane, whereas [3H]mesulergine and N-[3H]methylspiperone were used, in the presence of excess guanine nucleotide [guanosine 5'-O-(3-thiotriphosphate)], to define binding to the low-affinity state of the 5-HT2C and 5-HT2A receptors, respectively. Antagonists labeled the high- and low-affinity states of each receptor with comparable affinities. Previously identified inverse agonists of the 5-HT2C receptor behaved as silent antagonists in our systems even when the receptor was overexpressed at a relatively high density. In contrast, the ability of agonists to bind differentially to the high- and low-affinity states of the 5-HT2A and 5-HT2C receptors was highly correlated (r2 = 0.86 and 0.96, respectively) with their intrinsic activities. These data suggest that high-affinity agonist states can account for agonist efficacy at human 5-HT2A or 5-HT2C receptors without the need for considering additional transition or active states of the receptor-ligand complex. The procedure described herein may expedite drug discovery efforts by predicting intrinsic activities of ligands solely from ligand binding assays.

Interactions with 5-HT2A and 5-HT2C receptors may be important for the actions of atypical antipsychotic drugs, such as clozapine (CLOZ). In this study we characterized the interaction of chlorpromazine (CPZ) and three putative atypical antipsychotic drugs, risperidone (RIS), amperozide (AMP), and ORG 5222 (ORG) with the 5-HT2A and the 5-HT2C receptor. CLOZ was used as a reference agent. These agents had 5-HT2C receptor-binding affinities (Ki values) in the following rank order: ORG (0.9 nM) > CLOZ (13.2 nM) ≥ CPZ (27.1 nM) > RIS (112 nM) > > AMP (2580 nM). RIS (1.9 nM) and AMP (75.6 nM) had clearly higher affinities for the 5-HT2A than the 5-HT2C receptor; otherwise the 5-HT2A and 5-HT2C receptor affinities were approximately the same. Phosphoinositide hydrolysis studies in the rat choroid plexus revealed that all these agents were 5-HT2C receptor antagonists, with an approximately similar rank order of potency compared to the 5-HT2C receptor-binding data. Quantitative receptor autoradiography was used to study the regulation of 5-HT2A and 5-HT2C receptors after chronic treatment (14 days, SC injections once a day) with CLOZ (25 mg/kg), CPZ (15 mg/kg), RIS (0.3 mg/kg), AMP (5 mg/kg), and ORG (0.1 mg/kg). In the doses used, CLOZ, CPZ, and ORG decreased the frontal cortical 5-HT2A receptor binding of [3H]ketanserin and [125I]DOI by 40% to 60%. AMP also significantly decreased 5-HT2A receptor [3H]ketanserin binding by 30%, whereas RIS did not affect 5-HT2A receptor binding. In contrast to 5-HT2A receptors, only CLOZ significantly (by about 50%) decreased 5-HT2C receptor [3H]mesulergine and [125I]DOI binding in the choroid plexus. For comparison, CPZ was the only drug to significantly upregulate striatal D2 receptor-binding sites, whereas none of the drugs affected striatal D1 receptors. The main finding in this study is that 5-HT2A and 5-HT2C receptors are differentially regulated after chronic treatment with CLOZ, CPZ, RIS, AMP, and ORG.

Differential regulation of rat 5-ht2a and 5-ht2c receptors after chronic treatment with clozapine, chlorpromazine and three putative atypical antipsychotic drugs

Novel psychoplastogen DM506 reduces cue-induced heroin-seeking and inhibits tonic GABA currents in the Prelimbic Cortex

The 5-HT receptor subtypes 5-HT2A and 5-HT2C are important neurotherapeutic targets, though, obtaining selectivity over 5-HT2B and H1 receptors is challenging. Here, we delineated molecular determinants of selective binding to 5-HT2A and 5-HT2C receptors for novel 4-phenyl-2-dimethylaminotetralins (4-PATs). We synthesized 42 novel 4-PATs with halogen or aryl moieties at the C(4)-phenyl meta-position. Affinity, function, molecular modeling and 5-HT2A receptor mutagenesis studies were performed to understand structure-activity relationships at 5-HT2 -type and H1 receptors. Lead 4-PAT-type 5-HT2A /5-HT2C receptor inverse agonists were compared with pimavanserin, a selective 5-HT2A /5-HT2C receptor inverse agonist approved to treat Parkinson's disease-related psychosis, in the mouse head twitch response and locomotor activity assays, models relevant to antipsychotic drug development. Most 4-PAT diastereomers in the (2S,4R)-configuration bound non-selectively to 5-HT2A , 5-HT2C and H1 receptors, with >100-fold selectivity over 5-HT2B receptors, whereas diastereomers in the (2R,4R)-configuration bound preferentially to 5-HT2A over 5-HT2C receptors and had >100-fold selectivity over 5-HT2B and H1 receptors. Results suggest that G2385.42 and V2355.39 in 5-HT2A receptors (conserved in 5-HT2C receptors) are important for high affinity binding, whereas interactions with T1945.42 and W1584.56 determine H1 receptor affinity. The 4-PAT analog (2S,4R)-4-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, (2S,4R)-2k, a potent and selective 5-HT2A /5-HT2C receptor inverse agonist, had activity like pimavanserin in the mouse head twitch response assay but was distinct in not suppressing locomotor activity. The novel 4-PAT chemotype can yield selective 5-HT2A /5-HT2C receptor inverse agonists for antipsychotic drug development by optimizing ligand-receptor interactions in transmembrane domain 5. Chirality can be exploited to attain selectivity over H1 receptors, which may circumvent sedative effects.