Publications by authors named "Clinton E Canal"

30 Publications

  • Page 1 of 1

Evaluation of lorcaserin as an anticonvulsant in juvenile Fmr1 knockout mice.

Epilepsy Res 2021 09 27;175:106677. Epub 2021 May 27.

Mercer University, College of Pharmacy, Department of Pharmaceutical Sciences, 3001 Mercer University Drive, Atlanta, GA, 30341, USA. Electronic address:

Recent preclinical and clinical studies suggest that lorcaserin, a preferential serotonin 2C receptor (5-HTR) agonist that was approved for the treatment of obesity, possesses antiepileptic properties. Here, we tested whether lorcaserin (1, 3, 5.6, 10 mg/kg) is prophylactic against audiogenic seizures (AGSs) in juvenile Fmr1 knockout mice, a mouse model of fragile X syndrome (FXS). MPEP (30 mg/kg), a non-competitive mGluR5 receptor antagonist, was used as a positive control. As lorcaserin likely engages 5-HTRs at therapeutic doses, we pretreated one group of mice with the selective 5-HTR antagonist/inverse agonist, M100907 (0.03 mg/kg), alone or before administering lorcaserin (5.6 mg/kg), to discern putative contributions of 5-HTRs to AGSs. We also assessed lorcaserin's in vitro pharmacology at human (h) and mouse (m) 5-HTRs and 5-HTRs and its in vivo interactions at m5-HTRs and m5-HTRs. MPEP significantly decreased AGS prevalence (P = 0.011) and lethality (P = 0.038). Lorcaserin, 3 mg/kg, attenuated AGS prevalence and lethality by 14 % and 32 %, respectively, however, results were not statistically significant (P = 0.5 and P = 0.06); other doses and M100907 alone or with lorcaserin also did not significantly affect AGSs. Lorcaserin exhibited full efficacy agonist activity at h5-HTRs and m5-HTRs, and near full efficacy agonist activity at h5-HTRs and m5-HTRs; selectivity for activation of 5-HTRs over 5-HTRs was greater for human (38-fold) compared to mouse (13-fold) receptors. Lorcaserin displayed relatively low affinities at antagonist-labeled 5-HTRs and 5-HTRs, regardless of species. Lorcaserin (3 and 5.6 mg/kg) increased the 5-HTR-dependent head-twitch response (HTR) elicited by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) in mice (P = 0.03 and P = 0.02). At 3 mg/kg, lorcaserin alone did not elicit an HTR. If mice were treated with the selective 5-HTR antagonist SB 242084 (0.5 or 1 mg/kg) plus lorcaserin (3 mg/kg), a significantly increased HTR was observed, relative to vehicle (P = 0.01 and P = 0.03), however, the HTR was much lower than what was elicited by DOI or DOI plus lorcaserin. Lorcaserin, 3 mg/kg, significantly reduced locomotor activity on its own, an effect reversed by SB 242084, and lorcaserin also dose-dependently reduced locomotor activity when administered prior to DOI (Ps<0.002). These data suggest that lorcaserin may engage 5-HTRs as well as 5-HTRs in mice at doses as low as 3 mg/kg. The similar activity at m5-HTRs and m5-HTRs suggests careful dosing of lorcaserin is necessary to selectively engage 5-HTRs in vivo. In conclusion, lorcaserin was ineffective at preventing AGSs in Fmr1 knockout mice. Lorcaserin may not be a suitable pharmacotherapy for seizures in FXS.
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http://dx.doi.org/10.1016/j.eplepsyres.2021.106677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8296307PMC
September 2021

NeuroChat with Professor Clinton E. Canal.

ACS Chem Neurosci 2020 11;11(21):3485-3487

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http://dx.doi.org/10.1021/acschemneuro.0c00565DOI Listing
November 2020

()-5-(2'-Fluorophenyl)-,-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, a Serotonin Receptor Modulator, Possesses Anticonvulsant, Prosocial, and Anxiolytic-like Properties in an Knockout Mouse Model of Fragile X Syndrome and Autism Spectrum Disorder.

ACS Pharmacol Transl Sci 2020 Jun 21;3(3):509-523. Epub 2020 Feb 21.

Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University Health Sciences Center, Mercer University, 3001 Mercer University Drive, Atlanta, Georgia 30341, United States.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disabilities and a plethora of neuropsychiatric symptoms. FXS is the leading monogenic cause of autism spectrum disorder (ASD), which is defined clinically by repetitive and/or restrictive patterns of behavior and social communication deficits. Epilepsy and anxiety are also common in FXS and ASD. Serotonergic neurons directly innervate and modulate the activity of neurobiological circuits altered in both disorders, providing a rationale for investigating serotonin receptors (5-HTRs) as targets for FXS and ASD drug discovery. Previously we unveiled an orally active aminotetralin, ()-5-(2'-fluorophenyl)--dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), that exhibits partial agonist activity at 5-HTRs, 5-HTRs, and 5-HTRs and that reduces repetitive behaviors and increases social approach behavior in wild-type mice. Here we report that in an knockout mouse model of FXS and ASD, FPT is prophylactic for audiogenic seizures. No FPT-treated mice displayed audiogenic seizures, compared to 73% of vehicle-treated mice. FPT also exhibits anxiolytic-like effects in several assays and increases social interactions in both knockout and wild-type mice. Furthermore, FPT increases c-Fos expression in the basolateral amygdala, which is a preclinical effect produced by anxiolytic medications. Receptor pharmacology assays show that FPT binds competitively and possesses rapid association and dissociation kinetics at 5-HTRs and 5-HTRs, yet has slow association and rapid dissociation kinetics at 5-HTRs. Finally, we reassessed and report FPT's affinity and function at 5-HTRs, 5-HTRs, and 5-HTRs. Collectively, these observations provide mounting support for further development of FPT as a pharmacotherapy for common neuropsychiatric symptoms in FXS and ASD.
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http://dx.doi.org/10.1021/acsptsci.9b00101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296548PMC
June 2020

Structure-Activity Relationship Study of Psychostimulant Synthetic Cathinones Reveals Nanomolar Antagonist Potency of α-Pyrrolidinohexiophenone at Human Muscarinic M Receptors.

ACS Chem Neurosci 2020 03 3;11(6):960-968. Epub 2020 Mar 3.

Department of Pharmaceutical Sciences, Mercer University College of Pharmacy, 3001 Mercer University Drive, Atlanta, Georgia 30341, United States.

Synthetic cathinones (SCs) are designer, psychostimulant drugs of abuse that primarily act on monoamine transporters; little is known about their off-target liability. Abuse of pyrrolidine-containing SCs, such as α-PHP, has been linked to clinical features, including tachycardia and hypertension, and psychiatric events, including delusions and memory impairments-effects mimicking deliriant hallucinogens that are acetylcholine muscarinic receptor (MR) antagonists. α-PHP and nine analogs with modifications in the α-carbon side chain length and/or containing a methylenedioxy moiety were screened for activity at each of the five human MRs. Increasing the length of the α-carbon side chain of 1-phenyl-2-(pyrrolidin-1-yl)ethan-1-one analogs from a methyl (α-PPP) to a propyl (α-PVP) group caused a steep increase in affinity at all MR subtypes, and one extra carbon (α-PHP) further enhanced MR affinity; the presence of a methylenedioxy moiety generally hindered this effect. Highest MR affinity was observed with α-PHP at MRs-its MR affinity ( = 251 nM) was 302-fold higher than α-PPP's. MR-cAMP inhibition and β-arrestin recruitment assays showed that α-PHP is an MR antagonist ( = 120 and 502 nM, respectively). Additional experiments showed α-PHP is also an antagonist of MR-inositol phosphate production ( = 1.4 μM). Human toxicology studies report blood concentrations of pyrrolidine-containing SCs, including α-PHP, that reach micromolar levels during intoxication, indicating α-PHP's MR activity might have physiological relevance. As MRs and MRs are widely expressed in the autonomic and central nervous systems, α-PHP's anticholinergic activity might be relevant to adverse events associated with α-PHP intoxication.
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http://dx.doi.org/10.1021/acschemneuro.0c00008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104417PMC
March 2020

Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT and 5-HT G protein-coupled receptor affinity, 3D-QSAR and molecular modeling.

Bioorg Med Chem 2020 02 12;28(3):115262. Epub 2019 Dec 12.

Center for Drug Discovery, Department of Pharmaceutical Sciences, Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, USA.

The serotonin 5-HT G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure-affinity relationship (3D-QSAR) at the human 5-HT receptor for comparison with similar studies at the highly homologous 5-HT receptor. We report 35 new 5-SAT ligands, some with very high affinity (K ≤ 1 nM) and stereoselectivity at 5-HT + or 5-HT receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT, and, several ligands with high selectivity (up to 40-fold) at the 5-HT receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT over 5-HT receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT vs. 5-HT GPCRs, as may be required for successful clinical translation.
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http://dx.doi.org/10.1016/j.bmc.2019.115262DOI Listing
February 2020

M100907 and BD 1047 attenuate the acute toxic effects of methamphetamine.

Neurotoxicology 2019 09 1;74:91-99. Epub 2019 Jun 1.

Department of Pharmaceutical Sciences, Mercer University College of Pharmacy, Mercer University Health Sciences Center, Atlanta, GA, USA. Electronic address:

There are no Food and Drug Administration approved pharmacotherapies for methamphetamine (METH) overdose, thus identifying novel drug targets to prevent this devastating adverse event is a public-health imperative. Previous research suggests that serotonin and sigma receptors may contribute to the adverse effects of METH. The present study assessed whether pretreatment with the 5-HT receptor antagonist M100907 or the sigma 1 (σ) receptor antagonist BD 1047 attenuated METH-induced lethality, hyperthermia, convulsions, and seizures. Male, Swiss-Webster mice received intraperitoneal injections of M100907 (1 and 10 mg/kg), BD 1047 (10 mg/kg), or a combination of M100907 (1 mg/kg) and BD 1047 (10 mg/kg) prior to treatment with METH (78 mg/kg). Convulsions and lethality were assessed by observation, core body temperature was assessed by surgically implanted telemetric probes, and seizures were assessed by electroencephalography. M100907 reduced METH-elicited lethality from 67% to 33%, BD1047 reduced METH-elicited lethality from 67% to 50%, and combined administration of both agents eliminated lethality in all mice tested. Similarly, both agents and their combination reduced METH-elicited seizures and convulsions. None of the treatments decreased METH-induced hyperthermia. This research suggests that reducing METH-induced seizures is an important factor in reducing lethality associated with METH overdose. However, future studies should examine whether M100907 and BD 1047 modulate METH-induced hypertension and other adverse effects that may also contribute to METH overdose. Our data support the continued investigation of compounds that target 5-HT and σ receptors in METH-induced overdose, including their potential to yield emergency reversal agents.
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http://dx.doi.org/10.1016/j.neuro.2019.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750996PMC
September 2019

The synthetic cathinone psychostimulant α-PPP antagonizes serotonin 5-HT receptors: In vitro and in vivo evidence.

Drug Test Anal 2019 Jul 22;11(7):990-998. Epub 2019 Apr 22.

Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, Atlanta, GA, USA.

Synthetic cathinones (SCs) are β-keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5-HT receptors (5-HT R) and muscarinic M receptors (M R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M R (minimal displacement of [~K ] [ H]scopolamine up to 10 μM). However, two SCs, α-pyrrolidinopropiophenone (α-PPP) and 4-methyl-α-PPP, had low μM K values at 5-HT R. In 5-HT R-phosphoinositide hydrolysis assays, α-PPP and 4-methyl-α-PPP displayed inverse agonist activity. We further assessed the 5-HT R functional activity of α-PPP, and observed it competitively antagonized 5-HT R signaling stimulated by the 5-HT R agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI; K  = 851 nM). To assess in vivo 5-HT R activity, we examined the effects of α-PPP on the DOI-elicited head-twitch response (HTR) in mice. α-PPP dose-dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α-PPP. To corroborate a 5-HT R mechanism, we also tested 3,4-methylenedioxy-α-PPP (MDPPP) and 3-bromomethcathinone (3-BMC), SCs that we observed had 5-HT R K s > 10 μM. Neither MDPPP nor 3-BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α-PPP has antagonist interactions at 5-HT R in vitro that may translate at physiologically-relevant doses in vivo. Considering 5-HT R antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α-PPPs unpopularity compared to other monoamine transporter inhibitors.
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http://dx.doi.org/10.1002/dta.2582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615953PMC
July 2019

Ligand-directed serotonin 5-HT receptor desensitization and sensitization.

Eur J Pharmacol 2019 Apr 25;848:131-139. Epub 2019 Jan 25.

Center for Drug Discovery, Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, United States; Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, United States; Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, United States. Electronic address:

Exposure of G protein-coupled receptors (GPCRs) to agonists can desensitize receptor signaling and lead to drug tolerance, whereas inverse agonists can sensitize signaling. For example, activation of serotonin 5-HT GPCRs is pharmacotherapeutic for obesity, but there is tolerance to the anorectic effect of the only approved 5-HT agonist, lorcaserin. We tested the hypothesis that different agonists or inverse agonists differentially desensitize or sensitize, respectively, canonical 5-HT-mediated activation of phospholipase C (PLC) signaling in vitro. Lorcaserin, which displays potency and efficacy equal to 5-HT, desensitized the 5-HT receptor significantly more than 5-HT (p<0.05). Agonist chemotypes such as 2-aminotetralins, with similar potency but lower efficacy than 5-HT, produced little 5-HT desensitization. The piperazine agonist 1-(3-chlorophenyl)piperazine (mCPP), with lower potency but similar efficacy as 5-HT, elicited desensitization indistinguishable from 5-HT, while the piperazine agonist aripiprazole, with lower potency and efficacy, did not desensitize 5-HT-PLC signaling. Several 5-HT agonists also were assessed for β-arrestin recruitment-lorcaserin was a 'super-agonist', but a 2-aminotetralin and aripiprazole had nil activity, suggesting they are biased towards 5-HT-PLC signaling. We observed robust positive correlations between the magnitude of 5-HT desensitization and agonist efficacy to stimulate PLC or to recruit β-arrestin. In contrast, different inverse agonists caused different magnitudes of 5-HT sensitization that did not correlate with efficacy (or potency) to inhibit constitutive 5-HT-PLC signaling.  Assessment of the 5-HT-S407A point-mutated receptor indicated this residue's involvement in ligand-dependent desensitization, but we did not observe a role for protein kinase C.These data show that ligand structure uniquely impacts 5-HT desensitization and sensitization processes..
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http://dx.doi.org/10.1016/j.ejphar.2019.01.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767919PMC
April 2019

Effects of the second-generation "bath salt" cathinone alpha-pyrrolidinopropiophenone (α-PPP) on behavior and monoamine neurochemistry in male mice.

Psychopharmacology (Berl) 2019 Mar 1;236(3):1107-1117. Epub 2018 Oct 1.

Department of Pharmaceutical Sciences, Mercer University College of Pharmacy, Mercer University Health Sciences Center, Atlanta, GA, USA.

Rationale: Synthetic cathinones ("bath salts") are β-ketone analogs of amphetamines, yet few studies have examined their potential neurotoxic effects.

Objective: In the current study, we assessed the persistent behavioral and neurochemical effects of exposure to the second-generation synthetic cathinone α-pyrrolidinopropiophenone (α-PPP).

Methods: Male, Swiss-Webster mice were exposed to α-PPP (80 mg/kg) using a binge-like dosing regimen (QID, q2h). Behavior was assessed 4-5 days after the dosing regimen, and neurochemistry was assessed the following day. Behavior was studied using the elevated plus maze, Y-maze, and novel object recognition tests. Regional levels of dopamine, serotonin, norepinephrine, and the major dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in the prefrontal cortex and striatum using high-pressure liquid chromatography. Additional experiments assessed the time courses of the effects of α-PPP on locomotor activity and core temperature using telemetry.

Results: Exposure to α-PPP significantly impaired performance in the Y-maze, decreased overall exploratory activity in the novel object recognition test, and resulted in regionally specific depletions in monoamine neurochemistry. In contrast, it had no significant effect on elevated plus maze performance or object discrimination in the novel object recognition test. The locomotor-stimulant effects of α-PPP were comparable to cocaine (30 mg/kg), and α-PPP (80 mg/kg) did not induce hyperthermia.

Conclusions: α-PPP exposure results in persistent changes in exploratory behavior, spatial working memory, and monoamine neurochemistry. This research highlights potential dangers of α-PPP, including potential neurotoxicity, and suggests that the mechanisms underlying the persistent untoward effects of the cathinones may be distinct from those of the amphetamines.
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http://dx.doi.org/10.1007/s00213-018-5044-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443494PMC
March 2019

Serotonergic Psychedelics: Experimental Approaches for Assessing Mechanisms of Action.

Authors:
Clinton E Canal

Handb Exp Pharmacol 2018;252:227-260

Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, Atlanta, GA, USA.

Recent, well-controlled - albeit small-scale - clinical trials show that serotonergic psychedelics, including psilocybin and lysergic acid diethylamide, possess great promise for treating psychiatric disorders, including treatment-resistant depression. Additionally, fresh results from a deluge of clinical neuroimaging studies are unveiling the dynamic effects of serotonergic psychedelics on functional activity within, and connectivity across, discrete neural systems. These observations have led to testable hypotheses regarding neural processing mechanisms that contribute to psychedelic effects and therapeutic benefits. Despite these advances and a plethora of preclinical and clinical observations supporting a central role for brain serotonin 5-HT receptors in producing serotonergic psychedelic effects, lingering and new questions about mechanisms abound. These chiefly pertain to molecular neuropharmacology. This chapter is devoted to illuminating and discussing such questions in the context of preclinical experimental approaches for studying mechanisms of action of serotonergic psychedelics, classic and new.
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http://dx.doi.org/10.1007/164_2018_107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136989PMC
June 2019

Classics in Chemical Neuroscience: Aripiprazole.

ACS Chem Neurosci 2017 06 13;8(6):1135-1146. Epub 2017 Apr 13.

Department of Pharmaceutical Sciences, Center for Drug Discovery, Northeastern University , Boston, Massachusetts 02115, United States.

Aripiprazole was the first antipsychotic developed to possess agonist properties at dopamine D autoreceptors, a groundbreaking strategy that presented a new vista for schizophrenia drug discovery. The dopamine D receptor is the crucial target of all extant antipsychotics, and all developed prior to aripiprazole were D receptor antagonists. Extensive blockade of these receptors, however, typically produces extrapyramidal (movement) side effects, which plagued first-generation antipsychotics, such as haloperidol. Second-generation antipsychotics, such as clozapine, with unique polypharmacology and D receptor binding kinetics, have significantly lower risk of movement side effects but can cause myriad additional ones, such as severe weight gain and metabolic dysfunction. Aripiprazole's polypharmacology, characterized by its unique agonist activity at dopamine D and D and serotonin 5-HT receptors, as well as antagonist activity at serotonin 5-HT receptors, translates to successful reduction of positive, negative, and cognitive symptoms of schizophrenia, while also mitigating risk of weight gain and movement side effects. New observations, however, link aripiprazole to compulsive behaviors in a small group of patients, an unusual side effect for antipsychotics. In this review, we discuss the chemical synthesis, pharmacology, pharmacogenomics, drug metabolism, and adverse events of aripiprazole, and we present a current understanding of aripiprazole's neurotherapeutic mechanisms, as well as the history and importance of aripiprazole to neuroscience.
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http://dx.doi.org/10.1021/acschemneuro.7b00087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495458PMC
June 2017

The serotonin 5-HT receptor and the non-addictive nature of classic hallucinogens.

J Psychopharmacol 2017 01 15;31(1):127-143. Epub 2016 Nov 15.

2 Department of Pharmaceutical Sciences, Mercer University College of Pharmacy, Mercer University Health Sciences Center, Atlanta, USA.

Classic hallucinogens share pharmacology as serotonin 5-HT, 5-HT, and 5-HT receptor agonists. Unique among most other Schedule 1 drugs, they are generally non-addictive and can be effective tools in the treatment of addiction. Mechanisms underlying these attributes are largely unknown. However, many preclinical studies show that 5-HT agonists counteract the addictive effects of drugs from several classes, suggesting this pharmacological property of classic hallucinogens may be significant. Drawing from a comprehensive analysis of preclinical behavior, neuroanatomy, and neurochemistry studies, this review builds rationale for this hypothesis, and also proposes a testable, neurobiological framework. 5-HT agonists work, in part, by modulating dopamine neuron activity in the ventral tegmental area-nucleus accumbens (NAc) reward pathway. We argue that activation of 5-HT receptors on NAc shell, GABAergic, medium spiny neurons inhibits potassium Kv1.x channels, thereby enhancing inhibitory activity via intrinsic mechanisms. Together with experiments that show that addictive drugs, such as cocaine, potentiate Kv1.x channels, thereby suppressing NAc shell GABAergic activity, this hypothesis provides a mechanism by which classic hallucinogen-mediated stimulation of 5-HT receptors could thwart addiction. It also provides a potential reason for the non-addictive nature of classic hallucinogens.
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http://dx.doi.org/10.1177/0269881116677104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445387PMC
January 2017

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT Receptor Underlying the Pharmacology of Distinct Ligands.

ACS Chem Neurosci 2017 01 10;8(1):28-39. Epub 2016 Oct 10.

Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.

While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT receptors. In HEK293 cells expressing human 5-HT receptors, for example, (-)-trans-3'-Br-PAT and (-)-trans-3'-Cl-PAT are agonists regarding Gα-inositol phosphate signaling, whereas (-)-trans-3'-CF-PAT is an inverse agonist. To investigate the ligand-receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (K) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (K) of the antagonist radioligand [H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.
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http://dx.doi.org/10.1021/acschemneuro.6b00124DOI Listing
January 2017

An Orally Active Phenylaminotetralin-Chemotype Serotonin 5-HT7 and 5-HT1A Receptor Partial Agonist that Corrects Motor Stereotypy in Mouse Models.

ACS Chem Neurosci 2015 Jul 8;6(7):1259-70. Epub 2015 Jun 8.

Stereotypy (e.g., repetitive hand waving) is a key phenotype of autism spectrum disorder, Fragile X and Rett syndromes, and other neuropsychiatric disorders, and its severity correlates with cognitive and attention deficits. There are no effective treatments, however, for stereotypy. Perturbation of serotonin (5-HT) neurotransmission contributes to stereotypy, suggesting that distinct 5-HT receptors may be pharmacotherapeutic targets to treat stereotypy and related neuropsychiatric symptoms. For example, preclinical studies indicate that 5-HT7 receptor activation corrects deficits in mouse models of Fragile X and Rett syndromes, and clinical trials for autism are underway with buspirone, a 5-HT1A partial agonist with relevant affinity at 5-HT7 receptors. Herein, we report the synthesis, in vitro molecular pharmacology, behavioral pharmacology, and pharmacokinetic parameters in mice after subcutaneous and oral administration of (+)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine ((+)-5-FPT), a new, dual partial agonist targeting both 5-HT7 (Ki = 5.8 nM, EC50 = 34 nM) and 5-HT1A (Ki = 22 nM, EC50 = 40 nM) receptors. Three unique, heterogeneous mouse models were used to assess the efficacy of (+)-5-FPT to reduce stereotypy: idiopathic jumping in C58/J mice, repetitive body rotations in C57BL/6J mice treated with the NMDA antagonist, MK-801, and repetitive head twitching in C57BL/6J mice treated with the 5-HT2 agonist, DOI. Systemic (+)-5-FPT potently and efficaciously reduced or eliminated stereotypy in each of the mouse models without altering locomotor behavior on its own, and additional tests showed that (+)-5-FPT, at the highest behaviorally active dose tested, enhanced social interaction and did not cause behaviors indicative of serotonin syndrome. These data suggest that (+)-5-FPT is a promising medication for treating stereotypy in psychiatric disorders.
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http://dx.doi.org/10.1021/acschemneuro.5b00099DOI Listing
July 2015

Novel 4-substituted-N,N-dimethyltetrahydronaphthalen-2-amines: synthesis, affinity, and in silico docking studies at serotonin 5-HT2-type and histamine H1 G protein-coupled receptors.

Bioorg Med Chem 2015 Apr 7;23(7):1588-600. Epub 2015 Feb 7.

Department of Medicinal Chemistry and Department of Chemistry, University of Florida, Gainesville, FL 32610-0485, United States; Center for Drug Discovery, Department of Pharmaceutical Sciences and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115-5000, United States. Electronic address:

Syntheses were undertaken of derivatives of (2S,4R)-(-)-trans-4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (4-phenyl-2-dimethylaminotetralin, PAT), a stereospecific agonist at the serotonin 5-HT2C G protein-coupled receptor (GPCR), with inverse agonist activity at 5-HT2A and 5-HT2B GPCRs. Molecular changes were made at the PAT C(4)-position, while preserving N,N-dimethyl substitution at the 2-position as well as trans-stereochemistry, structural features previously shown to be optimal for 5-HT2 binding. Affinities of analogs were determined at recombinant human 5-HT2 GPCRs in comparison to the phylogenetically closely-related histamine H1 GPCR, and in silico ligand docking studies were conducted at receptor molecular models to help interpret pharmacological results and guide future ligand design. In most cases, C(4)-substituted PAT analogs exhibited the same stereoselectivity ([-]-trans>[+]-trans) as the parent PAT across 5-HT2 and H1 GPCRs, albeit, with variable receptor selectivity. 4-(4'-substituted)-PAT analogs, however, demonstrated reversed stereoselectivity ([2S,4R]-[+]-trans>[2S,4R]-[-]-trans), with absolute configuration confirmed by single X-ray crystallographic data for the 4-(4'-Cl)-PAT analog. Pharmacological affinity results and computational results herein support further PAT drug development studies and provide a basis for predicting and interpreting translational results, including, for (+)-trans-4-(4'-Cl)-PAT and (-)-trans-4-(3'-Br)-PAT that were previously shown to be more potent and efficacious than their corresponding enantiomers in rodent models of psychoses, psychostimulant-induced behaviors, and compulsive feeding ('binge-eating').
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http://dx.doi.org/10.1016/j.bmc.2015.01.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363177PMC
April 2015

A novel aminotetralin-type serotonin (5-HT) 2C receptor-specific agonist and 5-HT2A competitive antagonist/5-HT2B inverse agonist with preclinical efficacy for psychoses.

J Pharmacol Exp Ther 2014 May 21;349(2):310-8. Epub 2014 Feb 21.

Center for Drug Discovery (C.E.C., R.G.B.), Department of Pharmaceutical Sciences (C.E.C., R.G.B.), and Department of Chemistry and Chemical Biology (R.G.B.), Northeastern University, Boston, Massachusetts; Department of Psychiatry (D.M.), Medicinal Chemistry (D.F., R.S., K.K., R.G.B.), and Psychology (N.E.R., K.L.R.), University of Florida, Gainesville, Florida.

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (-)-trans-(2S,4R)-4-(3'[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (-)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (-)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (-)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (-)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (-)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (-)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders.
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http://dx.doi.org/10.1124/jpet.113.212373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989798PMC
May 2014

Molecular pharmacology and ligand docking studies reveal a single amino acid difference between mouse and human serotonin 5-HT2A receptors that impacts behavioral translation of novel 4-phenyl-2-dimethylaminotetralin ligands.

J Pharmacol Exp Ther 2013 Dec 30;347(3):705-16. Epub 2013 Sep 30.

Center for Drug Discovery (C.E.C., T.C.-S., Y.L., R.G.B.), Department of Pharmaceutical Sciences (C.E.C., T.C.-S., Y.L., R.G.B.), and Department of Chemistry and Chemical Biology (R.G.B.), Northeastern University, Boston, Massachusetts; and Department of Medicinal Chemistry (M.S.K., T.C.-S.) and Department of Psychiatry (D.M.), University of Florida, Gainesville, Florida.

During translational studies to develop 4-phenyl-2-dimethylaminotetralin (PAT) compounds for neuropsychiatric disorders, the (2R,4S)-trans-(+)- and (2S,4R)-trans-(-)-enantiomers of the analog 6-hydroxy-7-chloro-PAT (6-OH-7-Cl-PAT) demonstrated unusual pharmacology at serotonin (5-HT) 5-HT2 G protein-coupled receptors (GPCRs). The enantiomers had similar affinities (Ki) at human (h) 5-HT2A receptors (≈ 70 nM). In an in vivo mouse model of 5-HT2A receptor activation [(±)-(2,5)-dimethoxy-4-iodoamphetamine (DOI)-elicited head twitch], however, (-)-6-OH-7-Cl-PAT was about 5-fold more potent than the (+)-enantiomer at attenuating the DOI-elicited response. It was discovered that (+)-6-OH-7-Cl-PAT (only) had ≈ 40-fold-lower affinity at mouse (m) compared with h5-HT2A receptors. Molecular modeling and computational ligand docking studies indicated that the 6-OH moiety of (+)- but not (-)-6-OH-7-Cl-PAT could form a hydrogen bond with serine residue 5.46 of the h5-HT2A receptor. The m5-HT2A as well as m5-HT2B, h5-HT2B, m5-HT2C, and h5-HT2C receptors have alanine at position 5.46, obviating this interaction; (+)-6-OH-7-Cl-PAT also showed ≈ 50-fold lower affinity than (-)-6-OH-7-Cl-PAT at m5-HT2C and h5-HT2C receptors. Mutagenesis studies confirmed that 5-HT2A S5.46 is critical for (+)- but not (-)-6-OH-7-Cl-PAT binding, as well as function. The (+)-6-OH-7-Cl-PAT enantiomer showed partial agonist effects at h5-HT2A wild-type (WT) and m5-HT2A A5.46S point-mutated receptors but did not activate m5-HT2A WT and h5-HT2A S5.46A point-mutated receptors, or h5-HT2B, h5-HT2C, and m5-HT2C receptors; (-)-6-OH-7-Cl-PAT did not activate any of the 5-HT2 receptors. Experiments also included the (2R,4S)-trans-(+)- and (2S,4R)-trans-(-)-enantiomers of 6-methoxy-7-chloro-PAT to validate hydrogen bonding interactions proposed for the corresponding 6-OH analogs. Results indicate that PAT ligand three-dimensional structure impacts target receptor binding and translational outcomes, supporting the hypothesis that GPCR ligand structure governs orthosteric binding pocket molecular determinants and resulting pharmacology.
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http://dx.doi.org/10.1124/jpet.113.208637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836315PMC
December 2013

Molecular Determinants for Ligand Binding at Serotonin 5-HT and 5-HT GPCRs: Experimental Affinity Results Analyzed by Molecular Modeling and Ligand Docking Studies.

Int J Quantum Chem 2012 Dec;112(24):3807-3814

Department of Medicinal Chemistry. College of Pharmacy, University of Florida, Gainesville, Florida 32610.

Ligands that activate the serotonin 5-HT G protein-coupled receptor (GPCR) may be therapeutic for psychoses, addiction, and other neuropsychiatric disorders. Ligands that are antagonists at the closely related 5-HT GPCR also may treat neuropsychiatric disorders; in contrast, 5-HT activation may cause hallucinations. 5-HT-specific agonist drug design is challenging because 5-HT GPCRs share 80% transmembrane (TM) homology, same second messenger signaling, and no crystal structures are reported. To help delineate molecular determinants underlying differential binding and activation of 5-HT GPCRs, 5-HT, and 5-HT homology models were built from the -adrenergic GPCR crystal structure and equilibrated in a lipid phosphatidyl choline bilayer performing molecular dynamics simulations. Ligand docking studies at the 5-HT receptor models were conducted with the (2, 4)- and (2, 4)-enantiomers of the novel 5-HT agonist/5-HT antagonist -4-phenyl--dimethyl-2-aminotetralin (PAT) and its 4'-chlorophenyl congners. Results indicate PAT-5-HT molecular interactions especially in TM domain V are important for the (2, 4) enantiomer, whereas, TM domain VI and VII interactions are more important for the (2, 4) enantiomer.
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http://dx.doi.org/10.1002/qua.24237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3729958PMC
December 2012

Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4-iodoamphetamine elicited head-twitch response model.

Neuropharmacology 2013 Jul 23;70:112-21. Epub 2013 Jan 23.

University of Florida, Department of Medicinal Chemistry, PO Box 1000485, 1600 SW Archer Road, Gainesville, FL 32610-0485, USA.

There are seemingly conflicting data in the literature regarding the role of serotonin (5-HT) 5-HT2C receptors in the mouse head-twitch response (HTR) elicited by the hallucinogenic 5-HT2A/2B/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Namely, both 5-HT2C receptor agonists and antagonists, regarding 5-HT2C receptor-mediated Gq-phospholipase C (PLC) signaling, reportedly attenuate the HTR response. The present experiments tested the hypothesis that both classes of 5-HT2C receptor compounds could attenuate the DOI-elicited-HTR in a single strain of mice, C57Bl/6J. The expected results were considered in accordance with ligand functional selectivity. Commercially-available 5-HT2C agonists (CP 809101, Ro 60-0175, WAY 161503, mCPP, and 1-methylpsilocin), novel 4-phenyl-2-N,N-dimethyl-aminotetralin (PAT)-type 5-HT2C agonists (with 5-HT2A/2B antagonist activity), and antagonists selective for 5-HT2A (M100907), 5-HT2C (SB-242084), and 5-HT2B/2C (SB-206553) receptors attenuated the DOI-elicited-HTR. In contrast, there were differential effects on locomotion across classes of compounds. The 5-HT2C agonists and M100907 decreased locomotion, SB-242084 increased locomotion, SB-206553 resulted in dose-dependent biphasic effects on locomotion, and the PATs did not alter locomotion. In vitro molecular pharmacology studies showed that 5-HT2C agonists potent for attenuating the DOI-elicited-HTR also reduced the efficacy of DOI to activate mouse 5-HT2C receptor-mediated PLC signaling in HEK cells. Although there were differences in affinities of a few compounds at mouse compared to human 5-HT2A or 5-HT2C receptors, all compounds tested retained their selectivity for either receptor, regardless of receptor species. Results indicate that 5-HT2C receptor agonists and antagonists attenuate the DOI-elicited-HTR in C57Bl/6J mice, and suggest that structurally diverse 5-HT2C ligands result in different 5-HT2C receptor signaling outcomes compared to DOI.
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http://dx.doi.org/10.1016/j.neuropharm.2013.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754837PMC
July 2013

Drug discovery targeting human 5-HT(2C) receptors: residues S3.36 and Y7.43 impact ligand-binding pocket structure via hydrogen bond formation.

Eur J Pharmacol 2011 Dec 12;673(1-3):1-12. Epub 2011 Oct 12.

Department of Medicinal Chemistry, University of Florida, P.O. Box 100485, 1600 SW Archer Road, Gainesville, FL 32610-0485, USA.

Specific activation of serotonin (5-HT) 5-HT(2C) G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. Mutagenesis coupled with computational and molecular modeling experiments based on the human β₂ adrenergic receptor structure was employed to delineate the interactions of different ligands at human 5-HT(2C) residues D3.32, S3.36 and Y7.43. No binding of the tertiary amine radioligand ([³H]-mesulergine) could be detected when the 5-HT(2C) D3.32 residue was mutated to alanine (D3.32A). The S3.36A point-mutation greatly reduced affinity of primary amine ligands, modestly reduced affinity of a secondary amine, and except for the 5-HT(2C)-specific agonist N(CH₃)₂-PAT, affinity of tertiary amines was unaffected. Molecular modeling results indicated that the primary amines form hydrogen bonds with the S3.36 residue, whereas, with the exception of N(CH₃)₂-PAT, tertiary amines do not interact considerably with this residue. The Y7.43A point-mutation greatly reduced affinity of 5-HT, yet reduced to a lesser extent the affinity of tryptamine that lacks the 5-hydroxy moiety present in 5-HT; modeling results indicated that the 5-HT 5-hydroxy moiety hydrogen bonds with Y7.43 at the 5-HT(2C) receptor. Additional modeling results showed that 5-HT induced a hydrogen bond between Y7.43 and D3.32. Finally, modeling results revealed two low-energy binding modes for 5-HT in the 5-HT(2C) binding pocket, supporting the concept that multiple agonist binding modes may stabilize different receptor active conformations to influence signaling. Ligand potencies for modulating WT and point-mutated 5-HT(2C) receptor-mediated phospholipase C activity were in accordance with the affinity data. Ligand efficacies, however, were altered considerably by the S3.36A mutation only.
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http://dx.doi.org/10.1016/j.ejphar.2011.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224194PMC
December 2011

Impact of RNA editing on functions of the serotonin 2C receptor in vivo.

Front Neurosci 2010 16;4:26. Epub 2010 Mar 16.

Center for Molecular Neuroscience, Vanderbilt University School of Medicine Nashville, TN, USA.

Transcripts encoding 5-HT(2C) receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT(2C-VGV), exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT(2C-VGV) receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT(2C-VGV) receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT(2C-VGV) receptors. However, enhanced behavioral sensitivity to a 5-HT(2C) receptor agonist was also seen in mice expressing 5-HT(2C-VGV) receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT(2C-VGV) receptors had greater sensitivity to a 5-HT(2C) inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT(2C) receptor binding sites in the brains of mice solely expressing 5-HT(2C-VGV) receptors. We conclude that 5-HT(2C-VGV) receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT(2C) receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT(2C) receptor binding sites in brain. We further caution that the pattern of 5-HT(2C) receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor.
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http://dx.doi.org/10.3389/neuro.23.001.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858556PMC
January 2011

The serotonin 2C receptor potently modulates the head-twitch response in mice induced by a phenethylamine hallucinogen.

Psychopharmacology (Berl) 2010 Apr 19;209(2):163-74. Epub 2010 Feb 19.

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.

Rationale: Hallucinogenic serotonin 2A (5-HT(2A)) receptor partial agonists, such as (+ or -)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT(2A) receptor antagonists. In addition to 5-HT(2A) receptors, DOI and most other serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT(2C) receptors.

Objectives: We tested for involvement of 5-HT(2C) receptors in the HTR induced by DOI.

Results: Comparison of 5-HT(2C) receptor knockout and wild-type littermates revealed an approximately 50% reduction in DOI-induced HTR in knockout mice. Also, pretreatment with either the 5-HT(2C) receptor antagonist SB206553 or SB242084 eradicated a twofold difference in DOI-induced HTR between the standard inbred mouse strains C57BL/6J and DBA/2J, and decreased the DOI-induced HTR by at least 50% in both strains. None of several measures of 5-HT(2A) receptors in frontal cortex explained the strain difference, including 5-HT(2A) receptor density, Galpha(q) or Galpha(i/o) protein levels, phospholipase C activity, or DOI-induced expression of Egr1 and Egr2. 5-HT(2C) receptor density in the brains of C57BL/6J and DBA/2J was also equivalent, suggesting that 5-HT(2C) receptor-mediated intracellular signaling or other physiological modulators of the HTR may explain the strain difference in response to DOI.

Conclusions: We conclude that the HTR to DOI in mice is strongly modulated by 5-HT(2C) receptor activity. This novel finding invites reassessment of hallucinogenic mechanisms involving 5-HT(2) receptors.
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http://dx.doi.org/10.1007/s00213-010-1784-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868321PMC
April 2010

RNA editing of the serotonin 2C receptor and expression of Galpha(q) protein: genetic mouse models do not support a role for regulation or compensation.

J Neurochem 2009 Mar 20;108(5):1136-42. Epub 2009 Jan 20.

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

The serotonin 2C (5-HT(2C)) receptor undergoes RNA editing at five bases in a region of the pre-mRNA encoding the second intracellular loop, generating many unique 5-HT(2C) receptor isoforms. Mechanisms regulating in vivo expression of different edited 5-HT(2C) receptor isoforms are poorly understood, as are the adaptive consequences of variation in editing profiles. Recent findings suggest a putative relationship between expression levels of Galpha(q/11) protein and the degree of editing of 5-HT(2C) receptor transcripts. To elucidate the potential regulatory or adaptive role of Galpha(q/11) protein levels, we quantified editing of 5-HT(2C) receptor RNA transcripts in Galpha(q) null mice and protein levels of Galpha(q) and Galpha(11) in transgenic male mice solely expressing either the non-edited (INI) or the fully edited (VGV) isoforms of the 5-HT(2C) receptor. Pyrosequencing of RNA isolated from amygdaloid cortex in Galpha(q) null and wild-type mice revealed no significant differences in 5-HT(2C) receptor mRNA editing profiles. Cortical tissue from INI/y, VGV/y, and wild-type mice was assayed for expression of Galpha(q) and Galpha(11) subunits by Western blotting. No differences in signal density between wild-type and INI/y or VGV/y groups were found, indicating equivalent levels of Galpha(q) and Galpha(11) protein. Together, these data do not support a causal or compensatory relationship between 5-HT(2C) receptor RNA editing and G(q) protein levels.
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http://dx.doi.org/10.1111/j.1471-4159.2008.05852.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742694PMC
March 2009

Intra-amygdala injections of CREB antisense impair inhibitory avoidance memory: role of norepinephrine and acetylcholine.

Learn Mem 2008 Sep 26;15(9):677-86. Epub 2008 Aug 26.

Neuroscience Program, University of Illinois, Urbana-Champaign, Illinois 61820, USA.

Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 microL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the beta-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala.
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http://dx.doi.org/10.1101/lm.904308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632786PMC
September 2008

Different temporal profiles of amnesia after intra-hippocampus and intra-amygdala infusions of anisomycin.

Behav Neurosci 2007 Aug;121(4):732-41

Neuroscience Program, University of Illinois, 603 East Daniel Street, Champaign, IL 61820, USA.

Systemic or intra-hippocampal administration of the protein synthesis inhibitor anisomycin generally leads to impairments in memory tested 24 hr or more after training but spares memory for a few hours after training. Thus, amnesia does not appear immediately after training but develops with time, findings most often interpreted as evidence for distinct short- and long-term memory processes. However, time courses for the onset of amnesia vary substantially after treatment with protein synthesis inhibitors. Some of the variability across experiments may reflect task-related differences or, perhaps relatedly, may reflect memory processing mediated by different neural systems. In the present experiments, anisomycin was infused into either the hippocampus or the amygdala 20 min before inhibitory avoidance training. Similar to previous findings, intra-hippocampus injections of anisomycin impaired memory tested 48 hr after training yet spared memory tested 4 hr after training. In contrast, intra-amygdala injections of anisomycin impaired memory tested at 0.5, 4, and 48 hr after training, revealing no evidence for spared memory at short times after training. The distinct temporal properties for amnesia following anisomycin injections into the hippocampus or amygdala may reflect different consequences for memory of perturbations of the neural system in which the manipulation is made.
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http://dx.doi.org/10.1037/0735-7044.121.4.732DOI Listing
August 2007

Amnesia produced by altered release of neurotransmitters after intraamygdala injections of a protein synthesis inhibitor.

Proc Natl Acad Sci U S A 2007 Jul 18;104(30):12500-5. Epub 2007 Jul 18.

Neuroscience Program, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA.

Amnesia produced by protein synthesis inhibitors such as anisomycin provides major support for the prevalent view that the formation of long-lasting memories requires de novo protein synthesis. However, inhibition of protein synthesis might disrupt other neural functions to interfere with memory formation. Intraamygdala injections of anisomycin before inhibitory avoidance training impaired memory in rats tested 48 h later. Release of norepinephrine (NE), dopamine (DA), and serotonin, measured at the site of anisomycin infusions, increased quickly by approximately 1,000-17,000%, far above the levels seen under normal conditions. NE and DA release later decreased far below baseline for several hours before recovering at 48 h. Intraamygdala injections of a beta-adrenergic receptor antagonist or agonist, each timed to blunt effects of increases and decreases in NE release after anisomycin, attenuated anisomycin-induced amnesia. In addition, similar to the effects on memory seen with anisomycin, intraamygdala injections of a high dose of NE before training impaired memory tested at 48 h after training. These findings suggest that altered release of neurotransmitters may mediate amnesia produced by anisomycin and, further, raise important questions about the empirical bases for many molecular theories of memory formation.
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http://dx.doi.org/10.1073/pnas.0705195104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1941498PMC
July 2007

Modulation of memory with septal injections of morphine and glucose: effects on extracellular glucose levels in the hippocampus.

Physiol Behav 2006 Feb 15;87(2):298-303. Epub 2005 Dec 15.

Section of Endocrinology, Yale University School of Medicine, USA.

The concentration of glucose in the extracellular fluid (ECF) of the hippocampus decreases substantially during memory testing on a hippocampus-dependent memory task. Administration of exogenous glucose, which enhances task performance, prevents this decrease, suggesting a relationship between hippocampal glucose availability and memory performance. In the present experiment, spontaneous alternation performance and task-related changes in hippocampal ECF glucose were assessed in rats after intraseptal administration of morphine, which impairs memory on a spontaneous alternation task, and after co-administration of intraseptal glucose, which attenuates that impairment. Consistent with previous findings, spontaneous alternation testing resulted in a decrease in hippocampal ECF glucose levels in control rats. However, rats that received intraseptal morphine prior to testing showed memory impairments and an absence of the task-related decrease in hippocampal ECF glucose levels. Intraseptal co-administration of glucose with morphine attenuated the memory impairment, and ECF glucose levels in the hippocampus decreased in a manner comparable to that seen in control rats. These data suggest that fluctuations in hippocampal ECF glucose levels may be a marker of mnemonic processing and support the view that decreases in extracellular glucose during memory testing reflect increased glucose demand during memory processing.
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http://dx.doi.org/10.1016/j.physbeh.2005.10.016DOI Listing
February 2006

Glucose injections into the dorsal hippocampus or dorsolateral striatum of rats prior to T-maze training: modulation of learning rates and strategy selection.

Learn Mem 2005 Jul-Aug;12(4):367-74. Epub 2005 Jul 18.

The Neuroscience Program,University of Illinois, Urbana-Champaign, IL 61820, USA.

The present experiments examined the effects of injecting glucose into the dorsal hippocampus or dorsolateral striatum on learning rates and on strategy selection in rats trained on a T-maze that can be solved by using either a hippocampus-sensitive place or striatum-sensitive response strategy. Percentage strategy selection on a probe trial (P(crit)) administered after rats achieved criterion (nine of 10 correct choices) varied by group. All groups predominately exhibited a response strategy on a probe trial administered after overtraining, i.e., after 90 trials. In experiment 1, rats that received intrahippocampal glucose injections showed enhanced acquisition of the T-maze and showed increased use of response solutions at P(crit) compared with that of unimplanted and artificial cerebral spinal fluid (aCSF)-treated groups. These findings suggest that glucose enhanced hippocampal functions to accelerate the rate of learning and the early adoption of a response strategy. In experiment 2, rats that received intrastriatal glucose injections exhibited place solutions early in training and reached criterion more slowly than did aCSF controls, with learning rates comparable to those of unoperated and operated-uninjected controls. Relative to unoperated, operated-uninjected and glucose-injected rats, rats that received intrastriatal aCSF injections showed enhanced acquisition of the T-maze and increased use of response solutions at P(crit). The unexpected enhanced acquisition seen after striatal aCSF injections suggests at least two possible interpretations: (1) aCSF impaired striatal function, thereby releasing competition with the hippocampus and ceding control over learning to the hippocampus during early training trials; and (2) aCSF enhanced striatal functioning to facilitate striatal-sensitive learning. With either interpretation, the results indicate that intrastriatal glucose injections compensated for the aCSF-induced effect. Finally, enhanced acquisition regardless of treatment was accompanied by rapid adoption of a response solution for the T-maze.
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http://dx.doi.org/10.1101/lm.88205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183254PMC
October 2005

Increases in extracellular fluid glucose levels in the rat hippocampus following an anesthetic dose of pentobarbital or ketamine-xylazine: an in vivo microdialysis study.

Physiol Behav 2005 Feb 22;84(2):245-50. Epub 2004 Dec 22.

The Neuroscience Program, University of Illinois, Champaign, IL 61820, USA.

Using in vivo microdialysis, we examined glucose levels in the extracellular fluid (ECF) of the hippocampus and in the blood prior to and during pentobarbital (45 mg/kg) or ketamine-xylazine (66 mg/kg, 7 mg/kg) anesthesia. Anesthesia with either pentobarbital or ketamine-xylazine significantly increased hippocampal ECF glucose levels (mean peak increases of +71% and +85%, respectively). In addition, there were substantial increases in blood glucose levels (mean peak increases of +24% and +30%, respectively). The increased levels of hippocampal ECF glucose during anesthesia complement past evidence for decreases in ECF glucose in the hippocampus observed while rats perform a memory task sensitive to hippocampal damage, providing further support for the view that ECF glucose levels in the hippocampus are dynamically coupled to local neural activity.
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http://dx.doi.org/10.1016/j.physbeh.2004.11.009DOI Listing
February 2005
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