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Biophysical validation of serotonin 5-HT2A and 5-HT2C receptor interaction.

ABSTRACT: The serotonin (5-HT) 5-HT2A receptor (5-HT2AR) and 5-HT2C receptor (5-HT2CR) in the central nervous system are implicated in a range of normal behaviors (e.g., appetite, sleep) and physiological functions (e.g., endocrine secretion) while dysfunctional 5-HT2AR and/or 5-HT2CR are implicated in neuropsychiatric disorders (e.g., addiction, obesity, schizophrenia). Preclinical studies suggest that the 5-HT2AR and 5-HT2CR may act in concert to regulate the neural bases for behavior. Here, we utilize three distinct biophysical and immunocytochemistry-based approaches to identify and study this receptor complex in cultured cells. Employing a split luciferase complementation assay (LCA), we demonstrated that formation of the 5-HT2AR:5-HT2CR complex exists within 50 nm, increases proportionally to the 5-HT2CR:5-HT2AR protein expression ratio, and is specific to the receptor interaction and not due to random complementation of the luciferase fragments. Using a proximity ligation assay (PLA), we found that cells stably expressing both the 5-HT2AR and 5-HT2CR exhibit 5-HT2AR:5-HT2CR heteroreceptor complexes within 40 nm of each other. Lastly, bioluminescence resonance energy transfer (BRET) analyses indicates the formation of a specific and saturable 5-HT2AR:5-HT2CR interaction, suggesting that the 5-HT2AR and 5-HT2CR form a close interaction within 10 nm of each other in intact live cells. The bioengineered receptors generated for the LCA and the BRET exhibit 5-HT-mediated intracellular calcium signaling as seen for the native receptors. Taken together, this study validates a very close 5-HT2AR:5-HT2CR interaction in cultured cells.

SUBMITTER: Felsing DE

PROVIDER: S-EPMC6114921 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Timing deficits are observed in patients with schizophrenia. Serotonergic hallucinogens can also alter the subjective experience of time. Characterizing the mechanism through which the serotonergic system regulates timing will increase our understanding of the linkage between serotonin (5-HT) and schizophrenia, and will provide insight into the mechanism of action of hallucinogens. We investigated whether interval timing in mice is altered by hallucinogens and other 5-HT2 receptor ligands. C57BL/6J mice were trained to perform a discrete-trials temporal discrimination task. In the discrete-trials task, mice were presented with two levers after a variable interval. Responding on lever A was reinforced if the interval was <6.5 s, and responding on lever B was reinforced if the interval was >6.5 s. A 2-parameter logistic function was fitted to the proportional choice for lever B (%B responding), yielding estimates of the indifference point (T50) and the Weber fraction (a measure of timing precision). The 5-HT2A antagonist M100907 increased T50, whereas the 5-HT2C antagonist SB-242,084 reduced T50. The results indicate that 5-HT2A and 5-HT2C receptors have countervailing effects on the speed of the internal pacemaker. The hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI; 3 mg/kg IP), a 5-HT2 agonist, flattened the response curve at long stimulus intervals and shifted it to the right, causing both T50 and the Weber fraction to increase. The effect of DOI was antagonized by M100907 (0.03 mg/kg SC) but was unaffected by SB-242,084 (0.1 mg/kg SC). Similar to DOI, the selective 5-HT2A agonist 25CN-NBOH (6 mg/kg SC) reduced %B responding at long stimulus intervals, and increased T50 and the Weber fraction. These results demonstrate that hallucinogens alter temporal perception in mice, effects that are mediated by the 5-HT2A receptor. It appears that 5-HT regulates temporal perception, suggesting that altered serotonergic signaling may contribute to the timing deficits observed in schizophrenia and other psychiatric disorders.

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Biophysical validation of serotonin 5-HT2A and 5-HT2C receptor interaction.

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