Hallucinogenic 5-HT2A receptor (5-HT2AR) agonists-induced head-twitch response (HTR) is regulated by Gs signaling pathway. Formation of heterodimers between 5-HT2AR and metabotropic glutamate mGlu2 receptor (mGluR2) is essential for the hallucinogenic 5-HT2AR agonist-induced HTR. In order to investigate the effects of mGluR2 agonists and inverse agonists on hallucinogenic 5-HT2AR agonists DOM-induced HTR, C57BL/6 mice were pretreated with mGluR2 agonists (LY379268, LY354740, LY404039) or the inverse agonist LY341495, and the HTR was manually counted after administering DOM immediately. IP-One (IP1) HTRF assay and cAMP assay were performed to evaluate the effect of LY341495 or LY354740 on DOM-induced Gq and Gs activation in Human Embryonic Kidney-293 (HEK-293) T-type cells co-expressing 5-HT2AR and mGluR2. The results showed that DOM-induced HTR in mice was dose-dependently inhibited by LY379268, LY354740, and LY404039, while it was dose-dependently enhanced by LY341495. Moreover, LY341495 reversed the inhibitory effect of LY354740 on DOM-induced HTR. In HEK-293T cells co-expressing 5-HT2AR and mGluR2, DOM-induced cAMP level was decreased by LY354740 and increased by LY341495, but DOM-induced IP1 level was not regulated by LY354740 or LY341495. The regulation of DOM-induced HTR by mGluR2 agonists and inverse agonists is closely related to 5-HT2AR-mediated Gs signaling pathway. In HEK-293T cells co-expressing 5-HT2AR and mGluR2 A677S/A681P/A685G mutant (mGluR2 3 A mutant), DOM-induced cAMP level was not regulated by LY354740, but was significantly enhanced by LY341495. The 5-HT2AR/mGluR2 heterodimers is critical for DOM-induced HTR and cAMP level, both of which are inhibited by mGluR2 agonists and enhanced by mGluR2 inverse agonists.

BACKGROUND: Serotonergic psychedelics exert their effects via their high affinity for serotonin (5-HT) receptors, particularly through activating 5-HT2A receptors (5-HT2AR), employing the frontal cortex-dependent head-twitch response (HTR). Although universally believed to be so, studies have not yet fully ascertained whether 5-HT2AR activation is the sole initiator of these psychedelic effects. This is because not all 5-HT2AR agonists exhibit similar pharmacologic properties.
OBJECTIVE: This study aims to identify and discriminate the roles of 5-HT2AR and 5-HT2CR in the HTR induced by Methallylescaline (MAL) and 4-Methyl-2,5,β-trimethoxyphenethylamine (BOD) in male mice. Also, an analysis of their potential neurotoxic properties was evaluated.
METHODS: Male mice treated with MAL and BOD were evaluated in different behavioral paradigms targeting HTR and neurotoxicity effects. Drug affinity, pharmacological blocking, and molecular analysis were also conducted to support the behavioral findings. The HTR induced by DOI has been extensively characterized in male mice, making it a good positive control for this study, specifically for comparing the pharmacological effects of our test compounds.
RESULTS: The activation of 5-HT2CR, alone or in concert with 5-HT2AR, produces a comparable degree of HTRs (at a dose of 1 mg·kg-1), with divergent 5-HT2CR- and 5-HT2AR-Gqα11-mediated signaling and enhanced neurotoxic properties (at a dose of 30 mg·kg-1) coupled with activated pro-inflammatory cytokines. These findings show these compounds\' potential psychedelic and neurotoxic effects in male mice.
CONCLUSIONS: These findings showed that while 5-HT2AR is the main initiator of HTR, the 5-HT2CR also has a distinct property that renders it effective in inducing HTR in male mice.

D3 receptors, a key component of the dopamine system, have emerged as a potential target of therapies to improve motor symptoms across neurodegenerative and neuropsychiatric conditions. In the present work, we evaluated the effect of D3 receptor activation on the involuntary head twitches induced by 2,5-dimethoxy-4-iodoamphetamine (DOI) at behavioral and electrophysiological levels. Mice received an intraperitoneal injection of either a full D3 agonist, WC 44 [4-(2-fluoroethyl)-N-[4-[4-(2-methoxyphenyl)piperazin 1-yl]butyl]benzamide] or a partial D3 agonist, WW-III-55 [N-(4-(4-(4-methoxyphenyl)piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide] five minutes before the intraperitoneal administration of DOI. Compared to the control group, both D3 agonists delayed the onset of the DOI-induced head-twitch response and reduced the total number and frequency of the head twitches. Moreover, the simultaneous recording of neuronal activity in the motor cortex (M1) and dorsal striatum (DS) indicated that D3 activation led to slight changes in a single unit activity, mainly in DS, and increased its correlated firing in DS or between presumed cortical pyramidal neurons (CPNs) and striatal medium spiny neurons (MSNs). Our results confirm the role of D3 receptor activation in controlling DOI-induced involuntary movements and suggest that this effect involves, at least in part, an increase in correlated corticostriatal activity. A further understanding of the underlying mechanisms may provide a suitable target for treating neuropathologies in which involuntary movements occur.

The head-twitch response (HTR) in mice is considered a behavioral model for hallucinogens and serotonin 5-HT2A receptor function, as well as Tourette syndrome in humans. It is mediated by 5-HT2A receptor agonists such as ( ±)- 2,5-dimethoxy-4-iodoamphetamine (DOI) in the prefrontal cortex (PFC). The 5-HT2A antagonist EMD 281014, can prevent both DOI-induced HTR during ageing and c-fos expression in different regions of PFC. Moreover, the nonselective monoamine releaser methamphetamine (MA) suppressed DOI-induced HTR through ageing via concomitant activation of inhibitory 5-HT1A receptors, but enhanced DOI-evoked c-fos expression. d-Fenfluramine is a selective 5-HT releaser and induces HTR in mice, whereas MA does not. Currently, we investigated whether EMD 281014 or MA would alter: (1) d-fenfluramine-induced HTR frequency in 20-, 30- and 60-day old mice, (2) d-fenfluramine-evoked c-fos expression in PFC, and (3) whether blockade of inhibitory serotonergic 5-HT1A- or adrenergic ɑ2-receptors would prevent suppressive effect of MA on d-fenfluramine-induced HTR.
EMD 281014 (0.001-0.05 mg/kg) or MA (0.1-5 mg/kg) blocked d-fenfluramine-induced HTR dose-dependently during ageing. The 5-HT1A antagonist WAY 100635 countered the inhibitory effect of MA on d-fenfluramine-induced HTR in 30-day old mice, whereas the adrenergic ɑ2 antagonist RS 79948 reversed MA\'s inhibitory effect in both 20- and 30- day old mice. d-Fenfluramine significantly increased c-fos expressions in PFC regions. MA (1 mg/kg) pretreatment significantly increased d-fenfluramine-evoked c-fos expression in different regions of PFC. EMD 281014 (0.05 mg/kg) failed to prevent d-fenfluramine-induced c-fos expression, but significantly increased it in one PFC region (PrL at - 2.68 mm).
EMD 281014 suppressed d-fenfluramine-induced HTR but failed to prevent d-fenfluramine-evoked c-fos expression which suggest involvement of additional serotonergic receptors in the mediation of evoked c-fos. The suppressive effect of MA on d-fenfluramine-evoked HTR is due to well-recognized functional interactions between stimulatory 5-HT2A- and the inhibitory 5-HT1A- and ɑ2-receptors. MA-evoked increases in c-fos expression in PFC regions are due to the activation of diverse monoaminergic receptors through increased synaptic concentrations of 5-HT, NE and/or DA, which may also account for the additive effect of MA on d-fenfluramine-evoked changes in c-fos expression. Our findings suggest potential drug receptor functional interaction during development when used in combination.

BACKGROUND: 4-Thio-substituted phenylalkylamines such as 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and 2,5-dimethoxy-4-n-propylthiophenethylamine (2C-T-7) produce psychedelic effects in humans and have been distributed as recreational drugs.
OBJECTIVE: The present studies were conducted to examine the structure-activity relationships (SAR) of a series of 4-thio-substituted phenylalkylamines using the head twitch response (HTR), a 5-HT2A receptor-mediated behavior induced by psychedelic drugs in mice. The HTR is commonly used as a behavioral proxy in rodents for human psychedelic effects and can be used to discriminate hallucinogenic and non-hallucinogenic 5-HT2A agonists.
METHODS: HTR dose-response studies with twelve different 4-thio-substituted phenylalkylamines were conducted in male C57BL/6 J mice. To detect the HTR, head movement was recorded electronically using a magnetometer coil and then head twitches were identified in the recordings using a validated method based on artificial intelligence.
RESULTS: 2C-T, the parent compound of this series, had relatively low potency in the HTR paradigm, but adding an α-methyl group increased potency fivefold. Potency was also increased when the 4-methylthio group was extended by one to three methylene units. Fluorination of the 4-position alkylthio chain, however, was detrimental for activity, as was the presence of a 4-allylthio substituent versus a propylthio group. 2C-T analogs containing a 4-benzylthio group showed little or no effect in the HTR paradigm, which is consistent with evidence that bulky 4-substituents can dampen agonist efficacy at the 5-HT2A receptor. Binding and functional studies confirmed that the compounds have nanomolar affinity for 5-HT2 receptor subtypes and act as partial agonists at 5-HT2A.
CONCLUSIONS: In general, there were close parallels between the HTR data and the known SAR governing activity of phenylalkylamines at the 5-HT2A receptor. These findings further support the classification of 2C-T compounds as psychedelic drugs.

The psychedelic 5-HT2A receptor (5HT2AR) agonist psilocybin (or the active metabolite psilocin) has emerged as potential useful drug for various neuropsychiatric diseases, with a rapid onset of therapeutic activity. However, the mechanisms responsible for such effects remain incompletely characterized. We aimed to study in vitro pharmacological profile and in vivo acute mechanism of psilocin/psilocybin. Competition binding studies with psilocin were performed in brain and cell cultures. The role of 5HT2AR, 5-HT2C receptors (5HT2CR) and 5-HT1A receptors (5HT1AR) on the psychosis-like head-twitch response (HTR) and on body temperature in mice after psilocybin administration were evaluated. Psilocin showed similar affinities for 5HT2AR (Ki: 120-173 nM), 5HT2CR (Ki: 79-311 nM) and 5-HT1AR (Ki: 152-146 nM) in human and mice brain. Psilocybin induced a dose-dependent HTR (maximal effect 17.07 ± 1.31 at 1 mg/kg i.p.) that was completely suppressed by the 5HT2AR antagonist MDL11939 (1 mg/kg). Higher doses of psilocybin (3 mg/kg) induced lower HTR (9.00 ± 0.53). The 5HT2CR antagonist SB242084 (0.1 mg/kg) increased HTR exerted by psilocybin (3 mg/kg). Psilocybin significantly raised core body temperature at low dose (0.125 mg/kg) (Emax=0.67 ± 0.15 °C), whereas a significant decrease was induced by doses over 1 mg/kg (Emax = -1.31 ± 0.16 °C). Pre-treatment with the 5HT1AR antagonist WAY100635 reversed the decrease of body temperature after psilocybin (1 mg/kg), causing hyperthermia (Emax = 0.94 ± 0.26 °C). The present work provides key findings on the 5HT2AR, 5-HT2CR and 5HT1AR involvement in the acute central effects of psilocybin. The results may be relevant for understanding the mechanism of action underlying the therapeutic effects and side effects of this psychedelic drug.

Classical psychedelics represent a subgroup of serotonergic psychoactive substances characterized by their distinct subjective effects on the human psyche. Another unique attribute of this drug class is that such effects become less apparent after repeated exposure within a short time span. The classification of psychedelics as a subgroup within the serotonergic drug family and the tolerance to their effects are replicated by the murine head twitch response (HTR) behavioral paradigm. Here, we aimed to assess tolerance and cross-tolerance to HTR elicited by psychedelic and nonpsychedelic serotonin 2A receptor (5-HT2AR) agonists in mice. We show that repeated (4 days) administration of the psychedelic 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induced a progressive decrease in HTR behavior. Tolerance to DOI-induced HTR was also observed 24 h after a single administration of this psychedelic. Pretreatment with the 5-HT2AR antagonist M100907 reduced not only the acute manifestation of DOI-induced HTR, but also the development of tolerance to HTR. Additionally, cross-tolerance became apparent between the psychedelics DOI and lysergic acid diethylamide (LSD), whereas repeated administration of the nonpsychedelic 5-HT2AR agonist lisuride did not affect the ability of these two psychedelics to induce HTR. At the molecular level, DOI administration led to down-regulation of 5-HT2AR density in mouse frontal cortex membrane preparations. However, development of tolerance to the effect of DOI on HTR remained unchanged in β-arrestin-2 knockout mice. Together, these data suggest that tolerance to HTR induced by psychedelics involves activation of the 5-HT2AR, is not observable upon repeated administration of nonpsychedelic 5-HT2AR agonists, and occurs via a signaling mechanism independent of β-arrestin-2.

Methamphetamine (MA) is a non-selective monoamine releaser and thus releases serotonin (5-HT), norepinephrine (NE) and dopamine (DA) from corresponding nerve terminals into synapses. DOI ((±)-2, 5-dimethoxy-4-iodoamphetamine) is a direct-acting serotonergic 5-HT2A/C receptor agonist and induces the head-twitch response (HTR) via stimulation of 5-HT2A receptor in mice. While more selective serotonin releasers such as d-fenfluramine evoke the HTR, monoamine reuptake blockers (e.g., cocaine) suppress the DOI-evoked HTR via indirect stimulation of serotonergic 5-HT1A- and adrenergic ɑ2-receptors. Since the induction of HTR by DOI is age-dependent, we investigated whether: (1) during development MA can evoke the HTR by itself, and (2) acute pretreatment with either the selective 5-HT2A receptor antagonist EMD 281014 or low-doses of MA can: (i) modulate the DOI-induced HTR in mice across postnatal days 20, 30 and 60, and (ii) alter the DOI-induced c-fos expression in mice prefrontal cortex (PFC). To further explore the possible modulatory effect of MA on DOI-induced HTR, we investigated whether blockade of inhibitory serotonergic 5-HT1A- or adrenergic ɑ2-receptors by corresponding selective antagonists (WAY 100635 or RS 79948, respectively), can prevent the effect of MA on DOI-induced HTR during aging.
Although neither EMD 281014 nor MA by themselves could evoke the HTR, acute pretreatment with either EMD 281014 (0.01, 0.05 and 0.1 mg/kg, i.p.) or MA (1, 2.5, 5 mg/kg, i.p.), dose-dependently suppressed the DOI-induced HTR across ages. While WAY 100635 significantly reversed the inhibitory effect of MA in 20- and 30-day old mice, RS 79948 failed to significantly counter MA\'s inhibitory effect. Moreover, DOI significantly increased c-fos expressions in several PFC regions. EMD 281014 prevented the DOI-induced increases in c-fos expression. Despite the inhibitory effect of MA on DOI-induced HTR, MA alone or in combination with DOI, significantly increased c-fos expression in several regions of the PFC.
The suppressive effect of MA on the DOI-evoked HTR appears to be mainly due to functional interactions between the HTR-inducing 5-HT2A receptor and the inhibitory 5-HT1A receptor. The MA-induced increase in c-fos expression in different PFC regions may be due to MA-evoked increases in synaptic concentrations of 5-HT, NE and/or DA.

Recent preclinical and clinical studies suggest that lorcaserin, a preferential serotonin 2C receptor (5-HT2CR) agonist that was approved for the treatment of obesity, possesses antiepileptic properties. Here, we tested whether lorcaserin (1, 3, 5.6, 10 mg/kg) is prophylactic against audiogenic seizures (AGSs) in juvenile Fmr1 knockout mice, a mouse model of fragile X syndrome (FXS). MPEP (30 mg/kg), a non-competitive mGluR5 receptor antagonist, was used as a positive control. As lorcaserin likely engages 5-HT2ARs at therapeutic doses, we pretreated one group of mice with the selective 5-HT2AR antagonist/inverse agonist, M100907 (0.03 mg/kg), alone or before administering lorcaserin (5.6 mg/kg), to discern putative contributions of 5-HT2ARs to AGSs. We also assessed lorcaserin\'s in vitro pharmacology at human (h) and mouse (m) 5-HT2CRs and 5-HT2ARs and its in vivo interactions at m5-HT2CRs and m5-HT2ARs. MPEP significantly decreased AGS prevalence (P = 0.011) and lethality (P = 0.038). Lorcaserin, 3 mg/kg, attenuated AGS prevalence and lethality by 14 % and 32 %, respectively, however, results were not statistically significant (P = 0.5 and P = 0.06); other doses and M100907 alone or with lorcaserin also did not significantly affect AGSs. Lorcaserin exhibited full efficacy agonist activity at h5-HT2CRs and m5-HT2CRs, and near full efficacy agonist activity at h5-HT2ARs and m5-HT2ARs; selectivity for activation of 5-HT2CRs over 5-HT2ARs was greater for human (38-fold) compared to mouse (13-fold) receptors. Lorcaserin displayed relatively low affinities at antagonist-labeled 5-HT2CRs and 5-HT2ARs, regardless of species. Lorcaserin (3 and 5.6 mg/kg) increased the 5-HT2AR-dependent head-twitch response (HTR) elicited by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) in mice (P = 0.03 and P = 0.02). At 3 mg/kg, lorcaserin alone did not elicit an HTR. If mice were treated with the selective 5-HT2CR antagonist SB 242084 (0.5 or 1 mg/kg) plus lorcaserin (3 mg/kg), a significantly increased HTR was observed, relative to vehicle (P = 0.01 and P = 0.03), however, the HTR was much lower than what was elicited by DOI or DOI plus lorcaserin. Lorcaserin, 3 mg/kg, significantly reduced locomotor activity on its own, an effect reversed by SB 242084, and lorcaserin also dose-dependently reduced locomotor activity when administered prior to DOI (Ps<0.002). These data suggest that lorcaserin may engage 5-HT2CRs as well as 5-HT2ARs in mice at doses as low as 3 mg/kg. The similar activity at m5-HT2CRs and m5-HT2ARs suggests careful dosing of lorcaserin is necessary to selectively engage 5-HT2CRs in vivo. In conclusion, lorcaserin was ineffective at preventing AGSs in Fmr1 knockout mice. Lorcaserin may not be a suitable pharmacotherapy for seizures in FXS.

BACKGROUND: Head-twitch response (HTR) is a manifestation of the serotonergic system behavioral pharmacology commonly used as a proxy of psychedelic drug action in rodents.
METHODS: We developed a minimally invasive magnetic ear tag reporter and designed a detection system that performs a comprehensive characterization of each potential HTR event on an electromagnetic readout.
RESULTS: Magnetic ear tags were easy to install and generally well tolerated by the animals. On the low-threshold first phase of detection, the tags\' signal recorded in a magnetometer was filtered and screened for potential HTR events. On the second phase, the detector performed a comprehensive spectral analysis evaluation of each event and identified the HTR characteristic distribution of power density. Our system delivered satisfactory performance in the identification of pharmacologically-induced HTR and discrimination power against common non-HTR behaviors.
METHODS: Our system offers a high-throughput solution for studying HTR in mice employing minimally invasive procedures and superior standalone discriminative power compared to our previously reported fully-automated approach.
CONCLUSIONS: High-throughput identification of HTR utilizing magnetic ear-tagging and biphasic detection delivers satisfactory detection and discrimination power employing less invasive procedures.