The serotonin 2C receptor potently modulates the head-twitch response in mice induced by a phenethylamine hallucinogen

Abstract

Rationale: Hallucinogenic serotonin 2A (5-HT(2A)) receptor partial agonists, such as (+ or -)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT(2A) receptor antagonists. In addition to 5-HT(2A) receptors, DOI and most other serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT(2C) receptors.

Objectives: We tested for involvement of 5-HT(2C) receptors in the HTR induced by DOI.

Results: Comparison of 5-HT(2C) receptor knockout and wild-type littermates revealed an approximately 50% reduction in DOI-induced HTR in knockout mice. Also, pretreatment with either the 5-HT(2C) receptor antagonist SB206553 or SB242084 eradicated a twofold difference in DOI-induced HTR between the standard inbred mouse strains C57BL/6J and DBA/2J, and decreased the DOI-induced HTR by at least 50% in both strains. None of several measures of 5-HT(2A) receptors in frontal cortex explained the strain difference, including 5-HT(2A) receptor density, Galpha(q) or Galpha(i/o) protein levels, phospholipase C activity, or DOI-induced expression of Egr1 and Egr2. 5-HT(2C) receptor density in the brains of C57BL/6J and DBA/2J was also equivalent, suggesting that 5-HT(2C) receptor-mediated intracellular signaling or other physiological modulators of the HTR may explain the strain difference in response to DOI.

Conclusions: We conclude that the HTR to DOI in mice is strongly modulated by 5-HT(2C) receptor activity. This novel finding invites reassessment of hallucinogenic mechanisms involving 5-HT(2) receptors.

Fig. 1

5-HT_2C receptor knockout male mice (N=7) exhibit significantly fewer head-twitch responses compared to littermate control mice (N=5) after 1 mg/kg DOI treatment

Fig. 2

a C57BL/6J and DBA/2J male mice exhibit a robust difference in head-twitch response to DOI (N=3 mice per dose level per strain). Dose–response profiles show a significant difference in maxima between strains. DBA/2J has approximately 1.5- to twofold greater head-twitch response than C57BL/6J. A 2×8 factorial analysis of variance reveals a significant strain by dose interaction that is resolved into differences between strains at dose levels of 0.4 mg/kg or higher, as indicated by stars. b DOI-induced head-twitch in C57BL/6J and DBA/2J mice is reduced but not eliminated by 5-HT_2C antagonist pretreatment, and the strain difference in head-twitch is eradicated by 5-HT_2C antagonist pretreatment (N=6 per group). The stars indicate both a significant reduction in head-twitch response after SB206553 or SB242084 pretreatment compared to saline pretreatment, and significant reduction in the difference between strains after 5-HT_2C antagonist pretreatment compared to saline (see text for details)

Fig. 3

EEDQ treatment inactivates 5-HT_2A receptor binding (N=6) in the mouse brain which is prevented by pretreatment with MDL100907 (0.25 mg/kg, N=4), but not SB206553 (3.0 mg/kg, N=4), suggesting that 3.0 mg/kg SB206553 does not bind to 5-HT_2A receptor binding sites at this dose or lower doses

Fig. 4

[¹²⁵I]-DOI autoradiography data showing 5-HT_2A and 5-HT_2C receptor high-affinity binding in cortical and striatal brain regions comparing six C57BL/6J mice (squares, N=6) and six DBA/2J mice (triangles, N=6). The y-axis is microcuries per gram of tissue, and the x-axis defines the brain regions anterior cingulate cortex (ACC), prefrontal cortex (PFC), somatosensory cortex (SS ctx), anterior striatum (ASTR), and posterior striatum (PSTR). a [¹²⁵I]-DOI 5-HT_2A receptor binding. b [¹²⁵I]-DOI 5-HT_2C receptor binding

Fig. 5

There were no detectable differences in densities of Gα_q/11 and Gα_i/o proteins in cortex of C57BL/6J and DBA/2J. Inset shows a representative photomicrograph of Western blots of frontal cortex from C57BL/6J and DBA/2J mice showing equivalent levels of Gα_q/11 and Gα_i/o proteins

Fig. 6

The figure plots the basal and stimulated counts connected by mouse for each strain. Despite induction of significant 5-HT-stimulated [³H]-inositol phosphate levels produced by phospholipase C in frontal cortex from C57BL/6J and DBA/2J mice (N=6 per strain), there were no differences between strains

Fig. 7

Interaction plots are shown for relative gene expression from (a) Egr1 and (b) Egr2 real-time polymerase chain reaction experiments. Means ± SEM for gene expression (y-axis) are shown against DOI condition (x-axis) and strain (white or black symbols, N=5 per group, 20 mice total). Egr1 and Egr2 expression in the frontal cortex of C57BL/6J and DBA/2J mice are equivalently increased by DOI, independent of a basal strain difference in gene expression; there is no indication of a strain difference