The 5-HT6 receptor has been implicated in a variety of cognitive processes including habitual behaviors, learning, and memory. It is found almost exclusively in the brain, is expressed abundantly in striatum, and localizes to neuronal primary cilia. Primary cilia are antenna-like, sensory organelles found on most neurons that receive both chemical and mechanical signals from other cells and the surrounding environment; however, the effect of 5-HT6 receptor function on cellular morphology has not been examined. We confirmed that 5-HT6 receptors were localized to primary cilia in wild-type (WT) but not 5-HT6 knockout (5-HT6KO) in both native mouse brain tissue and primary cultured striatal neurons then used primary neurons cultured from WT or 5-HT6KO mice to study the function of these receptors. Selective 5-HT6 antagonists reduced cilia length in neurons cultured from wild-type mice in a concentration and time-dependent manner without altering dendrites, but had no effect on cilia length in 5-HT6KO cultured neurons. Varying the expression levels of heterologously expressed 5-HT6 receptors affected the fidelity of ciliary localization in both WT and 5-HT6KO neurons; overexpression lead to increasing amounts of 5-HT6 localization outside of the cilia but did not alter cilia morphology. Introducing discrete mutations into the third cytoplasmic loop of the 5-HT6 receptor greatly reduced, but did not entirely eliminate, trafficking of the 5-HT6 receptor to primary cilia. These data suggest that blocking 5-HT6 receptor activity reduces the length of primary cilia and that mechanisms that regulate trafficking of 5-HT6 receptors to cilia are more complex than previously thought.

receptors increases the likelihood for receptors to localize outside of the primary cilium and have been associated with changes in cilia morphology and dendritic outgrowth. In the present study, we explore the role of 5-HT6R rescue on neuronal morphology in primary neuronal cultures from 5-HT6R-KO mice, while maintaining a more physiological level of expression, wherein the receptor localizes to cilia in 80-90% of neurons (similar to endogenous 5-HT6R localization). We found that rescue of 5-HT6R expression is sufficient to increase cilia length and dendritic outgrowth, but primarily in neurons in which the receptor is located exclusively in the primary cilia. Additionally, we found that expression of 5-HT6R mutants, deficient in agonist-stimulated cAMP or without the predicted Fyn kinase binding domain, maintain constitutive activity for stimulating cAMP and still increased the length of cilia, while the proposed Fyn kinase domain was required for stimulating dendritic outgrowth. These findings highlight the complexity of 5-HT6R function and localization, particularly when using exogenous overexpression, and provide greater understanding and potential mechanisms for 5-HT6R drug therapies.

Disruption of neuronal chloride ion (Cl À ) homeostasis has been linked to several pathological conditions, including substance use disorder, yet targeted pharmacotherapies are lacking. In this study, we explored the potential of serotonin 2A receptor (5-HT 2A R) agonism to reduce alcohol consumption in male wild-type C57Bl/6J mice and to ameliorate alcohol-induced inhibitory plasticity in the midbrain. We found that administration of the putative 5-HT 2A R agonist TCB-2 attenuated alcohol consumption and preference but did not alter water or saccharin consumption. We hypothesized that the selective behavioural effects of TCB-2 on alcohol drinking were due, at least in part, to effects of the agonist on ventral tegmental area (VTA) neurocircuitry. Alcohol consumption impairs Cl À transport in VTA GABA neurons, which acts as a molecular adaptation leading to increased alcohol self-administration. Using ex vivo electrophysiological recordings, we found that exposure to either intermittent volitional alcohol drinking or an acute alcohol injection diminished homeostatic Cl À transport in VTA GABA neurons. Critically, in vivo TCB-2 administration normalized Cl À transport in the VTA after alcohol exposure. Thus, we show a potent effect of alcohol consumption on VTA inhibitory circuitry, in the form of dysregulated Cl À homeostasis that is reversible with agonism of 5-HT 2A Rs. Our results provide insight into the potential therapeutic action of 5-HT 2A R agonists for alcohol abuse.

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