Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex

Abstract

Most neuropharmacological agents and many drugs of abuse modulate the activity of heptahelical G-protein-coupled receptors. Although the effects of these ligands result from changes in cellular signaling, their neurobehavioral activity may not correlate with results of in vitro signal transduction assays. 5-Hydroxytryptamine 2A receptor (5-HT2AR) partial agonists that have similar pharmacological profiles differ in the behavioral responses they elicit. In vitro studies suggest that different agonists acting at the same receptor may establish distinct patterns of signal transduction. Testing this hypothesis in the brain requires a global signal transduction assay that is applicable in vivo. To distinguish the cellular effects of the different 5-HT2AR agonists, we developed an assay for global signal transduction on the basis of high throughput quantification of rapidly modulated transcripts. Study of the responses to agonists in human embryonic kidney 293 cells stably expressing 5-HT2ARs demonstrated that each agonist elicits a distinct transcriptome fingerprint. We therefore studied behavioral and cortical signal transduction responses in wild-type and 5-HT2AR null-mutant mice. The hallucinogenic chemicals (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and lysergic acid diethylamide (LSD) stimulated a head-twitch behavioral response that was not observed with the nonhallucinogenic lisuride hydrogen maleate (LHM) and was absent in receptor null-mutant mice. We also found that DOI, LSD, and LHM each induced distinct transcriptome fingerprints in somatosensory cortex that were absent in 5-HT2AR null-mutants. Moreover, DOI and LSD showed similarities in the transcriptome fingerprints obtained that were not observed with the behaviorally inactive drug LHM. Our results demonstrate that chemicals acting at the 5-HT2AR induce specific cellular response patterns in vivo that are reflected in unique changes in the somatosensory cortex transcriptome.

Figure 1.

Quantitative transcriptional responses to 5-HT2AR agonists in 198A2 cells. Fold changes are expressed relative to vehicle-treated samples. A, Concentration-response curves for 5-HT, 5-methoxy-tryptamine (5-MeOT), tryptamine (TRYP), and DOI inducing the expression of egr-1 and cyr61. Data are means ± SEM from three samples. B, TFPs showing the pattern of response obtained for seven modulated transcripts. Gray indicates that agonist was not tested at that concentration.

Figure 2.

Behavioral responses to 5-HT2AR agonists. A, Chemical structure of the 5-HT2AR partial agonists studied in vivo. B, Wild-type (WT) and 5-HT2AR null-mutant (KO) mice (n = 6 per treatment group) were injected with vehicle DOI (2 mg/kg, i.p.), LSD (0.24 mg/kg, i.p.), or LHM (0.4 mg/kg, i.p.), and the head-twitch response was scored 20 min after injection for 30 min. ^***p < 0.001; Dunnett's post hoc test of one-way ANOVA of the normalized events.

Figure 3.

Characterization of the gene expression changes induced by DOI in mouse SSCx. A, Autoradiographs of coronal brain sections from mice injected with vehicle (n = 4) or DOI (8 mg/kg, i.p.; n = 8), killed after 60 min, and hybridized with cRNAs for c-fos or egr-1. DOI significantly increased the level of expression of genes assayed in SSCx (p < 0.05). B, Time courses of the DOI-induced gene response assayed by qRT-PCR. Mice were injected with DOI (2 mg/kg, i.p.; filled symbols; n = 4-8) or vehicle (n = 2-4; open symbols) and killed at the times indicated. Results represent mean ± SEM of the fold changes compared with basal levels of expression. C, Dose-response curves of the DOI-induced gene response assayed by qRT-PCR. Mice were injected intraperitoneally with the doses of DOI indicated (n = 6) and killed after 60 min. Data expressed are means ± SEM of the fold changes in comparison with levels of expression in vehicle-injected mice.

Figure 4.

TFPs induced by 5-HT2AR agonists in SSCx from wild-type (WT) and 5-HT2AR null-mutant (KO) mice determined by qRT-PCR assays. Mice were injected intraperitoneally with vehicle (WT, n = 12; KO, n = 6), DOI (2 mg/kg; WT; n = 21; KO, n = 6), LSD (0.24 mg/kg; WT, n = 12; KO, n = 6), or LHM (0.4 mg/kg; WT; n = 12; KO, n = 6) and killed after 60 min. Relative fold changes in levels of mRNA expression of 13 transcripts are shown. The basal expression levels of all of the assayed transcripts were comparable in WT and KO mice (data not shown) (Table 1). See also supplementary Table 3.