Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C 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-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor

The Need to Improve Reporting of the Pharmacological Action of New Molecules

The molecular characterization of bioactive molecules, for example, small molecules targeting G-protein-coupled receptors, is evolving in complexity, impacting the meaning of terms like "agonist", "antagonist", and "selective", which, in the absence of detailed definitions and scientific consensus, can be sources of confusion in the literature. We discuss this issue and offer straightforward solutions to it

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

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 μL) 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 β-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

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

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

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5‑HT_2C Receptor Underlying the Pharmacology of Distinct Ligands

While exploring the structure–activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT_2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT_2C receptors. In HEK293 cells expressing human 5-HT_2C‑INI receptors, for example, (−)-<i>trans</i>-3′-Br-PAT and (−)-<i>trans</i>-3′-Cl-PAT are agonists regarding Gα_q-inositol phosphate signaling, whereas (−)-<i>trans</i>-3′-CF₃-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_2C 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 (<i>K</i>_i) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC₅₀ functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (−)-<i>trans</i>-3′-CF₃-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (−)-<i>trans</i>-3′-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (<i>K</i>_D) of the antagonist radioligand [³H]­mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT_2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT_2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand

An Orally Active Phenylaminotetralin-Chemotype Serotonin 5‑HT₇ and 5‑HT_1A Receptor Partial Agonist That Corrects Motor Stereotypy in Mouse Models

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-HT_1A partial agonist with relevant affinity at 5-HT₇ receptors. Herein, we report the synthesis, <i>in vitro</i> molecular pharmacology, behavioral pharmacology, and pharmacokinetic parameters in mice after subcutaneous and oral administration of (+)-5-(2′-fluorophenyl)-<i>N</i>,<i>N</i>-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine ((+)-5-FPT), a new, dual partial agonist targeting both 5-HT₇ (<i>K</i>_i = 5.8 nM, EC₅₀ = 34 nM) and 5-HT_1A (<i>K</i>_i = 22 nM, EC₅₀ = 40 nM) receptors. Three unique, heterogeneous mouse models were used to assess the efficacy of (<i>+</i>)-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 (<i>+</i>)-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 (<i>+</i>)-5-FPT is a promising medication for treating stereotypy in psychiatric disorders