Serotonin, but not N-methyltryptamines, activates the serotonin 2A receptor via a ß-arrestin2/Src/Akt signaling complex in vivo

Abstract

Hallucinogens mediate many of their psychoactive effects by activating serotonin 2A receptors (5-HT(2A)R). Although serotonin is the cognate endogenous neurotransmitter and is not considered hallucinogenic, metabolites of serotonin also have high affinity at 5-HT(2A)R and can induce hallucinations in humans. Here we report that serotonin differs from the psychoactive N-methyltryptamines by its ability to engage a β-arrestin2-mediated signaling cascade in the frontal cortex. Serotonin and 5-hydroxy-L-tryptophan (5-HTP) induce a head-twitch response in wild-type (WT) mice that is a behavioral proxy for 5-HT(2A)R activation. The response in β-arrestin2 knock-out (βarr2-KO) mice is greatly attenuated until the doses are elevated, at which point, βarr2-KO mice display a head-twitch response that can exceed that of WT mice. Direct administration of N-methyltryptamines also produces a greater response in βarr2-KO mice. Moreover, the inhibition of N-methyltransferase blocks 5-HTP-induced head twitches in βarr2-KO mice, indicating that N-methyltryptamines, rather than serotonin, primarily mediate this response. Biochemical studies demonstrate that serotonin stimulates Akt phosphorylation in the frontal cortex and in primary cortical neurons through the activation of a β-arrestin2/phosphoinositide 3-kinase/Src/Akt cascade, whereas N-methyltryptamines do not. Furthermore, disruption of any of the components of this cascade prevents 5-HTP-induced, but not N-methyltryptamine-induced, head twitches. We propose that there is a bifurcation of 5-HT(2A)R signaling that is neurotransmitter and β-arrestin2 dependent. This demonstration of agonist-directed 5-HT(2A)R signaling in vivo may significantly impact drug discovery efforts for the treatment of disorders wherein hallucinations are part of the etiology, such as schizophrenia, or manifest as side effects of treatment, such as depression.

Figure 1.

High doses of serotonin and 5-HTP can induce a head-twitch response in the βarr2-KO mice. A–D, 5-HTP and serotonin induce a head-twitch response in WT and βarr2-KO mice. Time course of 5-HTP (200 mg/kg, i.p.)-induced head-twitch response (A) and 5-HTP dose–response curves in WT and βarr2-KO mice and blockade of 5-HTP-induced (200 mg/kg) responses (B) after a 10 min pretreatment with M100907 (M100; 0.05 mg/kg, i.p.). WT versus βarr2-KO within dose: *p < 0.05; **p < 0.001; within genotype: 200 mg/kg 5-HTP versus M100 + 200 mg/kg 5-HTP, ^##p < 0.001. Time course of 5-HT (40 μg, i.c.v.)-induced head-twitch response (C) and serotonin dose–response curves in WT and βarr2-KO mice and blockade of 5-HT-induced (40 μg, i.c.v.) responses (D) after a 10 min pretreatment with M100907 (0.05 mg/kg, i.p.). WT versus βarr2-KO at the same dose: *p < 0.05; **p < 0.001; within genotype: 40 μg of 5-HT versus M100 + 40 μg of 5-HT: ^##p < 0.001. E, F, Inhibition of MAO-A enhances 5-HTP potency in both genotypes. Time course of 5-HTP (100 mg/kg, i.p.)-induced head-twitch responses after a 1 h pretreatment with clorgyline (Clor; 1 mg/kg, i.p.) or clorgyline vehicle (Veh; 0.9% saline, i.p.) (E) and the sum of twitches induced over the 60 min observation period at two doses of 5-HTP (50 and 100 mg/kg, i.p.) in WT and βarr2-KO mice and blockade of clorgyline-enhanced 5-HTP-induced (100 mg/kg) responses after a 10 min pretreatment with M100907 (0.05 mg/kg, i.p.) (F). WT versus βarr2-KO at the same dose: ***p < 0.0001. Vehicle pretreatment versus clorgyline pretreatment within genotype: ^#p < 0.05, ^##p < 0.001, ^###p < 0.0001; within genotype: clorgyline + 100 mg/kg 5-HTP versus M100 + clorgyline + 100 mg/kg 5-HTP: ^#p < 0.05. Mean ± SEM are shown.

Figure 2.

An N-methyltransferase inhibitor eliminates 5-HTP-induced head twitches in βarr2-KO mice. Pretreatment with an N-methyltransferase inhibitor, MTZ, blocks 5-HTP-induced head-twitch response in the βarr2-KO mice and attenuates the response in WT mice. Time course analysis of head twitches induced by 200 mg/kg 5-HTP (intraperitoneally) (A) and the total number of twitches observed over 60 min after a 10 min pretreatment with MTZ (125 ng, i.c.v.) or vehicle (5 μl of dH₂O, i.c.v.) (B). WT versus βarr2-KO with the same treatment: ***p < 0.001. Vehicle pretreatment versus MTZ pretreatment within genotype: ^#p < 0.05, ^##p < 0.01. Mean ± SEM are shown.

Figure 3.

N-Methyltryptamines induce more head twitches in βarr2-KO mice than their WT littermates. βarr2-KO mice treated with either N-methylserotonin (N-Me-5-HT) (A–D) or 5-MeO-DMT (E–H) display significantly more head twitches than WT mice. Time course of head twitches induced by N-methylserotonin (20 μg, i.c.v.) (A) and dose–response curve for the total number of head twitches observed over 30 min (B). WT versus βarr2-KO: *p < 0.05, **p < 0.01. C, Pretreatment (10 min) with the 5-HT_2AR antagonist M100907 (M100; 0.05 mg/kg, i.p.), but not vehicle (0.02% Tween 80), blocks the N-methylserotonin (20 μg, i.c.v.)-induced head-twitch response in both genotypes. WT versus βarr2-KO: *p < 0.05; values for M100907 treatment were 0 ± 0. D, Pretreatment (10 min) of WT mice with the N-methyltransferase inhibitor MTZ (125 ng, i.c.v.) or vehicle (5 μl of dH₂O, i.c.v.) has no effect on the N-methylserotonin (20 μg, i.c.v.)-induced head-twitch response. E, F, Time course of head twitches induced by 5-MeO-DMT (10 mg/kg, i.p.) (E) and dose–response curve for the total number of head twitches observed over 30 min (F). WT versus βarr2-KO: *p < 0.05, ***p < 0.001. G, Pretreatment (10 min) with M100907 (0.05 mg/kg, i.p.), but not vehicle, blocks the 5-MeO-DMT (10 mg/kg, i.p.)-induced head-twitch response in both genotypes. WT versus βarr2-KO: *p < 0.05; vehicle versus M100 within genotype: ^##p < 0.01, ^###p < 0.0001. H, Pretreatment (10 min) of C57BL/6J mice with MTZ (125 ng, i.c.v.) or vehicle (5 μl of dH₂O, i.c.v.) has no effect on the 5-MeO-DMT-induced (10 mg/kg, i.p.) head-twitch response. Mean ± SEM are shown.

Figure 4.

5-HTP stimulates Akt phosphorylation and the association of β-arrestin2/Akt/Src complex with the 5-HT_2AR in the frontal cortex. A, Treatment with 5-HTP (100 mg/kg, i.p.) for 10 min induces Akt phosphorylation (P-Akt) in the frontal cortex of WT but not βarr2-KO mice. 5-MeO-DMT (10 mg/kg, i.p.) does not induce Akt phosphorylation in either genotype [total Akt (T-Akt)]. Vehicle versus 5-HTP: **p < 0.01. B, 5-HT_2AR immunoprecipitation reveals that 5-HTP treatment (100 mg/kg, i.p.) for 10 min decreases 5-HT_2AR association with PSD-95 but increases 5-HT_2AR associations with β-arrestin2, Src, and Akt. 5-HTP treatment has no effect on PSD-95, Src, or Akt coimmunoprecipitation with 5-HT_2AR in βarr2-KO mice. C, 5-MeO-DMT treatment (10 mg/kg, i.p. for 10 min) does not displace PSD-95 from the immunoprecipitated 5-HT_2AR, nor does it cause associations with β-arrestin2, Src, or Akt in either WT or βarr2-KO mice. A “no protein” control (NP; antibody + beads) is shown for each representative immunoblot. The mean ± SEM of the densitometric analysis is shown. IB, Immunoblot.

Figure 5.

Serotonin stimulates Akt phosphorylation in mouse cortical neurons. A, A 10 min 5-HT (1 μm) treatment induces Akt phosphorylation in WT but not βarr2-KO primary cortical cultures. N-Methylserotonin (N-Me-5-HT) and 5-MeO-DMT do not stimulate Akt phosphorylation in either genotype. Vehicle versus 5-HT: ***p < 0.001. B, M100907 pretreatment (M100; 10 nm for 15 min) inhibits Akt phosphorylation by serotonin (5; 1 μm for 10 min) compared with neurons that were pretreated with vehicle [M100 vehicle (Veh; Vehicle on abscissa) = 0.0001% DMSO; serotonin vehicle (V; Vehicle in the legend) = 2 μm ascorbate] for the same time period. Vehicle versus 5-HT: ***p < 0.001, Bonferroni's post hoc analysis. C, Time course studies reveal Akt phosphorylation after treatment with 1 μm serotonin (left) but not 5-MeO-DMT (right) in primary cortical cultures from WT mice, whereas βarr2-KO neurons do not show Akt activation with either agonist. WT versus βarr2-KO: **p < 0.01, ***p < 0.001, Bonferroni's post hoc analysis. D, Serotonin induces (5; 1 μm for 10 min) Akt phosphorylation compared with vehicle (V; 2 μm ascorbate) in βarr2-KO neurons transfected with Myc-tagged β-arrestin2 (βarr2-myc), whereas those neurons transfected with empty vector (Mock) do not. Vehicle versus 5-HT: *p < 0.05. E, Pretreatment (10 μm for 1 h) with the PI3K inhibitor [LY294002 (LY)] or the Src inhibitor (PP2) blocks Akt phosphorylation induced by serotonin (5; 1 μm for 10 min) in WT primary cortical neurons [inhibitor vehicle (Veh; Vehicle on abscissa) = 0.1% DMSO; serotonin vehicle (V; Vehicle in the legend) = 2 μm ascorbate). Vehicle versus 5-HT: ***p < 0.001. Representative blots and densitometric analysis are provided. Mean ± SEM are shown.

Figure 6.

Inhibition of PI3K, Src, or Akt attenuates 5-HTP-mediated but not N-methyltryptamine-mediated head twitches in mice. A, B, Inhibitors of PI3K (LY294002), Src (PP2), or Akt (AKTi) attenuated 5-HTP-induced head twitches in normal mice. Time course of head-twitch responses (A) and the total number of head twitches (B) observed in C57BL/6J mice that were pretreated for 10 min with either vehicle (Veh; 1% DMSO in dH₂O, i.c.v.) or LY294002 (LY; 125 ng, i.c.v.), PP2 (300 ng, i.c.v.), or AKTi (55 ng, i.c.v.) before 5-HTP administration (200 mg/kg, i.p.). Vehicle versus inhibitor: *p < 0.05. C, Pretreatment with AKTi (55 ng, i.c.v. for 10 min) has no effect on 5-MeO-DMT-induced (10 mg/kg, i.p.) head twitches in C57BL/6J mice compared with vehicle-pretreated (1% DMSO in dH₂O, i.c.v.) mice. Mean ± SEM are shown.

Figure 7.

Inhibition of PI3K, Src, or Akt has no affect on 5-HTP-induced head twitches in βarr2-KO mice. Inhibitors of PI3K (LY294002), Src (PP2), or Akt (AKTi) have no effect on 5-HTP-induced head twitches in βarr2-KO mice. Time course of head-twitch responses (A) and the total number of head twitches (B) observed in βarr2-KO mice that were pretreated for 10 min with either vehicle (Veh; 1% DMSO in dH₂O, i.c.v.) or LY294002 (LY; 125 ng, i.c.v.), PP2 (300 ng, i.c.v.), or AKTi (55 ng, i.c.v.) before 5-HTP administration (200 mg/kg, i.p.). Mean ± SEM are shown.

Figure 8.

5-HTP-induced head twitches in WT mice are blocked by combined inhibition of N-methyltransferase and Akt. Pretreatment with the Akt inhibitor (AKTi) or the N-methyltransferase inhibitor (MTZ) individually reduce 5-HTP-induced twitches in WT mice; coadministration of the inhibitors is additive. Time course (A) and total numbers (B) of head twitches observed in WT mice after 10 min pretreatment with either vehicle (Veh; 0.1% DMSO in dH₂O, i.c.v.), AKTi (55 ng, i.c.v.), or MTZ (125 ng, i.c.v.) alone, or AKTi and MTZ concurrently before 5-HTP administration (200 mg/kg, i.p.) Vehicle versus inhibitor: ^##p < 0.01, ^###p < 0.001. AKTi versus AKTi + MTZ: **p < 0.01; MTZ versus AKTi + MTZ: ***p < 0.001. Mean ± SEM are shown.

Figure 9.

Schematic representing 5-HT_2AR signaling in vivo. When serotonin levels are increased (by direct administration or by blocking the MAO-A route of metabolism to the inactive 5-HIAA using clorgyline), an elevation of psychoactive N-methyltryptamine metabolites can occur via N-methyltransferase (NMT) metabolism of serotonin and tryptamines. Serotonin and these psychoactive metabolites then differentially activate the 5-HT_2AR to produce the head-twitch response in mice. Left, Serotonin promotes disengagement of PSD-95 and recruitment of β-arrestin2, PI3K, Src, and Akt to the 5-HT_2AR. This leads to Akt phosphorylation, which can be prevented when any member of this scaffold is disrupted. Inhibition of the kinases or disruption of the scaffold by removal of β-arrestin2 reduces serotonin-mediated head twitches in WT mice by ∼50%. Right, N-Methyltryptamines at the 5-HT_2AR do not induce Akt phosphorylation and mediate head twitches in mice independent of β-arrestin2 and Akt, because neither kinase inhibition nor β-arrestin2 deletion blocks N-methyltryptamine-induced head twitches. Furthermore, β-arrestin2 appears to dampen the effect of N-methyltryptamines in the head-twitch response as the response to the N-methyltryptamines is enhanced in the βarr2-KO mice. All of these effects are mediated by the 5-HT_2AR as the antagonist M100907 blocks the response in its entirety. Finally, an inhibitor of N-methyltransferase (MTZ) prevents the β-arrestin2-independent head-twitch response that occurs after administration of high doses of serotonin, further indicating that this response is mediated by the N-methyltryptamines.