Head-twitch response

The head-twitch response, also sometimes known as wet dog shakes in rats, is a rapid side-to-side head movement that occurs in mice and rats in association with serotonin 5-HT_2A receptor activation. Serotonergic psychedelics like lysergic acid diethylamide and psilocybin consistently induce the HTR in rodents, whereas non-hallucinogenic serotonin 5-HT_2A receptor agonists do not. Because of this, the HTR is widely employed in scientific research as an animal behavioral model of hallucinogen effects and in the discovery of new psychedelic drugs.
The HTR is one of the only behavioral paradigms for assessment of psychedelic-like effects in animals, with the other most notable test being drug discrimination. However, the HTR is far less costly and time-consuming than drug discrimination and hence has become much more popular in recent years. Limitations of the HTR include the fact that various other drugs besides serotonin 5-HT_2A receptor agonists, such as NMDA receptor antagonists and muscarinic acetylcholine receptor antagonists, also induce the HTR, and certain indirect non-hallucinogenic serotonin 5-HT_2A receptor activators, like 5-hydroxytryptophan and serotonin releasers, induce the response as well. While a useful test, it should not be regarded as a definitive predictor of human psychedelic potential.
The HTR was first described as an effect of psychedelics in the mid-1950s. It was subsequently proposed as a behavioral test of psychedelic-like effects in 1967. The HTR became widely used as a test of psychedelic-like effects by the mid-2000s. Automated versions of the HTR test, allowing for high-throughput screening, were developed in the 2010s and 2020s.

Description

The HTR is a rapid, rhythmic side-to-side or rotational head movement that intermittently occurs in mice and rats in association with central serotonin 5-HT_2A receptor activation. In mice, each individual head movement lasts about 10milliseconds and each HTR consists of 5 to 11individual head movements. The HTR is spontaneous and irregularly occurring over the drug's duration. Head twitches also occur naturally in rodents but occur at low frequencies and are only rarely observed in non-stimulated animals. Drugs inducing the HTR cause the frequency to increase by many orders of magnitude above the spontaneous rate. Within a 10-minute period, between 4 and 68head twitches have been observed following administration of DOI, depending on the dose, enantiomer, and rodent species and strain. The head twitches produced by HTR-inducing drugs are identical to spontaneous head twitches and to touch-induced head twitches.
In rats, the HTR is also sometimes known instead as wet dog shakes. This is because the response in rats can involve more of the whole body instead of just the head shaking and can resemble the shaking of dogs coming out of water. On account of the preceding, the test has also been referred to as the head-twitch response/wet dog shake test.
Serotonin 5-HT_2A receptor agonists show an inverted U-shaped dose–response curve for induction of the HTR in terms of its frequency. Tolerance rapidly develops to the induction of the HTR with many but not all serotonergic psychedelics. More specifically, tolerance has been observed with LSD, DOB, DOI, 2C-T-7, 25CN-NBOH, and 5-MeO-AMT, but not with DPT or DiPT. Development of tolerance to the HTR and other serotonin 5-HT_2A receptor agonist effects in animals parallels the rapid development of tolerance to the hallucinogenic effects of many psychedelics in humans, including LSD, DOM, psilocybin, and mescaline, among others. Conversely, similarly to the HTR with DPT and DiPT, tolerance does not appear to develop to the psychedelic effects of DMT, ayahuasca, or 5-MeO-DMT in humans. However, more recent clinical studies employing DMT by continuous intravenous infusion have found rapid and moderate acute tolerance development with DMT. Time-dependent supersensitivity to the HTR in animals has also been reported, for instance with DOI.
The effective doses of numerous serotonergic psychedelics in producing the HTR have been reviewed as well as correlated with human psychedelic doses.
The HTR is also known to occur in rabbits and certain mole-like shrews such as the least shrew. Conversely, it does not occur in other species like primates or humans. However, HTR-like behaviors are also induced by psychedelics in certain other animal species, for instance cats and stump-tailed macaque monkeys. Other related behaviors to head twitches induced by serotonergic agents in animals include ear scratching in mice, limb jerks or flicks in cats, head bobs in rabbits, and body scratches. However, other behaviors induced by psychedelics may not be as reliable as the HTR. In addition, ear scratches appear to be mediated primarily by activation of the serotonin 5-HT_2C receptor rather than by activation of the serotonin 5-HT_2A receptor. On the other hand, psychedelic-induced head bobs in rabbits appear to be mediated specifically by central serotonin 5-HT_2A receptor activation.

Procedure

The HTR method is reliable, straightforward, and simple to perform in that it merely involves direct behavioral observation following drug administration. No animal training or expensive equipment are necessarily required. The HTR can be measured in a single animal or a group of animals and can be observed in real-time or via video-recording and later observation. It can also be recorded via a magnet attached to the head or ear.
DOI is the most commonly used psychedelic to induce the HTR. DOI and other psychedelics show a biphasic or inverted U-shaped dose–response curve in terms of HTR induction. For example, no HTR is observed at 0.1mg/kg DOI, maximal HTR is observed at 1 to 10mg/kg, and lesser HTR is observed at 3 to 20mg/kg in rodents. The doses can vary depending on the rodent species and strain. Hence, based on the preceding, proper drug dosing is important for induction of the HTR.
A drawback of the HTR assay is that manual observation can be very laborious and time-consuming. More recently however, semi- and fully-automated forms of the assay, notably allowing for the possibility of high-throughput screening, have been developed.

Mechanisms

The HTR produced by serotonergic psychedelics, which act as non-selective serotonin receptor agonists, appears to be mediated specifically by agonism of the serotonin 5-HT_2A receptor. Selective and non-selective serotonin 5-HT_2A antagonists, like volinanserin, can block production of the HTR by serotonergic psychedelics. Similarly, the HTR of psychedelics is absent in serotonin 5-HT_2A receptor knockout mice. Restoration of the serotonin 5-HT_2A receptor to cortical neurons in these knockout mice can restore the HTR. The intracellular signaling cascade activated by the serotonin 5-HT_2A to produce the HTR appears to be the G_q pathway. However, the cascades have not been conclusively determined, and other pathways, such as the G_s and β-arrestin2 pathways, have also been implicated in other studies.
The HTR is mediated by central serotonin 5-HT_2A receptor activation. Hence, if a given serotonin 5-HT_2A receptor agonist produces the HTR, this can be considered evidence that the drug is able to cross the blood–brain barrier and exert central effects. Activation of serotonin 5-HT_2A receptors in the medial prefrontal cortex, with layer V pyramidal neurons especially implicated and with subsequent release of glutamate in this area, may be the origin of the HTR. However, other brain areas have also been independently implicated. For example, local injection of serotonergic psychedelics into the claustrum produces wet dog shakes in rats as well. Serotonin 5-HT_2A and metabotropic glutamate mGlu₂ receptor heterodimeric complexes may or may not be important for induction of the HTR by psychedelics, with research findings in this area being conflicting.
The HTR is said to resemble a strong pinna reflex involving the whole head. The pinna reflex can be elicited by tactile stimulation, for example stimulation of the ear by a fine hair. In the case of the HTR however, the reflex occurs without tactile stimulation. The HTR induced by the serotonin precursor 5-hydroxytryptophan has been found to be sensitive to environmental interference by background noise and can be prevented by local anesthesia of the pinna. These findings suggest that the HTR might be due specifically to disturbances of auditory sensory processing, although more research is needed to confirm this possibility.
The reasons for the biphasic or inverted U-shaped dose–response curve with psychedelics are unknown. However, activation of serotonin 5-HT_2C and 5-HT_1A receptors at higher doses appears to at least partly be involved. In contrast to the HTR, frontal cortex inositol monophosphate turnover due to serotonin 5-HT_2A receptor activation, which is associated with the hallucinogenic effects of serotonergic psychedelics, has an asymptotic dose–response curve akin to a saturation curve. Based on these findings, it was suggested that psychedelics may continue to increase serotonin 5-HT_2A receptor activation at higher doses but that behavioral disruption limits the expression of the HTR.
Tolerance and tachyphylaxis to the HTR and/or other effects of serotonergic psychedelics may be mediated by serotonin 5-HT_2A receptor downregulation. LSD, psilocin, DOM, DOI, and DOB have all been found to reduce the density of brain serotonin 5-HT_2A receptors in animals in vivo and/or to desensitize the receptor in transfected cell lines, and this downregulation has been found to recover very slowly. LSD has also been specifically shown to reduce brain serotonin 5-HT_2A receptor signaling in animals. Conversely however, DMT, which is not associated with tolerance development in humans, did not desensitize the serotonin 5-HT_2A receptor in cell lines. Activation of the serotonin 5-HT_2A receptor β-arrestin2 pathway may mediate serotonin 5-HT_2A receptor internalization and tolerance. However, findings are conflicting, as β-arrestin2 knockout mice still showed tolerance to the HTR induced by DOI. It is also notable that, in contrast to most G protein-coupled receptors, serotonin 5-HT_2A receptor downregulation has been found to occur in response to both agonists and antagonists of the receptor. Besides serotonin 5-HT_2A receptor downregulation, tolerance to psychedelics may also develop due to adaptations in downstream glutamate receptors. An alternative possibility to serotonin 5-HT_2A receptor biased agonism is that the lack of tolerance with drugs like DMT may simply be due to their very short durations.