Article Figures & Data
Figures
- 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-HT2AR 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-HT2AR 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-HT2AR immunoprecipitation reveals that 5-HTP treatment (100 mg/kg, i.p.) for 10 min decreases 5-HT2AR association with PSD-95 but increases 5-HT2AR associations with β-arrestin2, Src, and Akt. 5-HTP treatment has no effect on PSD-95, Src, or Akt coimmunoprecipitation with 5-HT2AR 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-HT2AR, 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-HT2AR 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-HT2AR 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-HT2AR. 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-HT2AR 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-HT2AR 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.
Tables
Figure Treatment in mice Serotonergic dose and n values 1A,B 5-HTP (mg/kg, i.p.) ±M100 100 150 200 M100 + 200 WT, βarr2-KO n = 13, 8 n = 7, 5 n = 8, 7 n = 6, 6 1C,D 5-HT (μg, i.c.v.) ±M100 10 20 40 M100 + 40 WT, βarr2-KO n = 4, 5 n = 7, 5 n = 5, 6 n = 4, 4 1E,F Veh/Clor (±M100) + 5-HTP Veh +50 Veh +100 Clor +50 Clor +100 Clor + M100 +100 WT, βarr2-KO n = 6, 5 n = 6, 5 n = 5, 6 n = 9, 8 n = 4, 4 2A,B Veh/MTZ + 5-HTP Veh + 200 MTZ + 200 WT, βarr2-KO n = 4, 5 n = 4, 5 3A,B N-Me-5-HT (μg, i.c.v.) 10 20 40 WT, βarr2-KO n = 4, 4 n = 5, 5 n = 4, 4 3C Veh/M100 + N-Me-5-HT Veh + 20 M100 + 20 WT, βarr2-KO n = 4, 4 n = 4, 4 3D Veh/MTZ + N-Me-5-HT Veh + 20 MTZ + 20 WT n = 5 n = 4 3E,F 5-MeO-DMT (mg/kg, i.p.) 5 10 15 WT, βarr2-KO n = 9, 10 n = 10, 16 n = 5, 5 3G Veh/M100 + 5-MeO-DMT Veh + 10 M100 + 10 WT, βarr2-KO n = 5, 9 n = 5, 5 3H Veh/MTZ + 5-MeO-DMT Veh + 10 MTZ + 10 C57BL/6 n = 6 n = 4 6A,B Veh, LY, PP2, AKTi + 5-HTP Veh + 200 LY + 200 PP2 + 200 AKTi + 200 C57BL/6 n = 10 n = 5 n = 5 n = 5 6C Veh, AKTi + 5-MeO-DMT 10 AKTi + 200 C57BL/6 n = 6 n = 4 7A,B Veh, LY, PP2, AKTi + 5-HTP 200 LY + 200 PP2 + 200 AKTi + 200 βbarr2-KO n = 7 n = 6 n = 6 n = 6 8A,B Veh, AKTi, MTZ, AKTi + MTZ + 5-HTP 200 MTZ + 200 AKTi + 200 MTZ + AKTi +200 WT n = 11 n = 6 n = 5 n = 5 Veh, Vehicle; 5-HT, serotonin; M100, M100907; Clor, clorgyline; N-Me-5-HT, N-methylserotonin; LY, LY294002. Serotonergic dosing routes: 5-HTP, mg/kg, i.p.; 5-HT and N-methylserotonin, μg, i.c.v.; 5-MeO-DMT, mg/kg, i.p. For inhibitor and antagonist dosage information, see Materials and Methods.
