Headshake Response

The head-twitch response (HTR), also sometimes known as wet dog shakes (WDS) 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 (LSD) and psilocybin consistently induce the HTR in rodents. 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 (5-HTP) and serotonin releasers, induce the response as well.

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 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 (also known as the pinna reflex).

In rats, the HTR is also sometimes known instead as wet dog shakes (WDS). 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 (HTR/WDS) 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 (which contains DMT), or 5-MeO-DMT in humans. Time-dependent supersensitivity to the HTR in animals has also been reported, for instance with DOI.

The effective doses (ED₅₀) of numerous serotonergic psychedelics in producing the HTR have been reviewed as well as correlated with human psychedelic doses.

HTR-like behaviors are also induced by psychedelics in 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 and simple to perform in that it simply involves direct behavioral observation following drug administration. No animal training or expensive equipment are 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.

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 antagonist (M100907), 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.

Activation of serotonin 5-HT_2A receptors in the medial prefrontal cortex (mPFC), 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. 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 (5-HTP) has been found to be sensitive to environmental interference by background noise and can be prevented by local anesthesia of the pinna (outer part of the ear). 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.

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 (GPCRs), 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.

Scientific validity

Head twitches do not occur with psychedelics in humans and head twitches lack face validity as an animal behavioral proxy of psychedelic effects. In any case, it has been said that head twitches might be a behavioral response to sensory disturbances during hallucinogenic experiences. On the other hand, many drugs that are not hallucinogenic in humans also induce the HTR. Despite the preceding limitations, the assay has strong predictive validity for hallucinogenic effects of serotonin 5-HT_2A receptor agonists in humans. There is a strong correlation between the capacity of serotonergic psychedelics to induce head twitches in rodents and their reported potency in inducing hallucinogenic effects in humans. The HTR is easily quantifiable and there is high agreement in counts between independent observers. In addition, there is a low level of within-subject and between-subject variability in induction of the HTR in animals.

Exceptions

Psychedelics lacking head twitches in animals

There are few or no known examples of serotonergic psychedelics with hallucinogenic effects in humans that do not produce the HTR in animals. One of the only known instances, the LSD prodrug ALD-52 (1-acetyl-LSD), could be explained by species differences in metabolism. Other possible exceptions, including various 2C psychedelics like 2C-B, 2C-I, and 2C-D, as well as the phenylpiperazine TFMPP, may be explained by these agents having relatively low intrinsic activity at the serotonin 5-HT_2A receptor and by species differences in sensitivity to HTR elicitation by serotonin 5-HT_2A receptor partial agonists (mice being more sensitive than rats). Dimethyltryptamine (DMT) shows effects on the HTR in mice that are highly strain-dependent, including producing an HTR comparable to other psychedelics, producing an HTR that is much weaker than that of other psychedelics, or producing no HTR at all. These conflicting results may be due to rapid metabolism of DMT and/or other peculiarities of DMT in different species.

Non-psychedelics inducing head twitches

The HTR can be non-specific and can have false positives, with head twitches also produced by some drugs that do not act through serotonin 5-HT₂ receptors. Examples of these agents include NMDA receptor antagonists like phencyclidine (PCP), certain benzodiazepines and Z-drugs like estazolam, triazolam, and zopiclone, α₂-adrenergic receptor antagonist, muscarinic acetylcholine receptor antagonist and scopolamine, serotonin 5-HT_1A receptor antagonists like WAY-100635 and UH-301, and CB₁ receptor antagonist. In the cases of benzodiazepines, rimonabant, and serotonin 5-HT_1A receptor antagonists however, this effect appeared to be mediated by indirect or direct activation of serotonin 5-HT_2A receptors. A number of other drugs, including the acetylcholine receptor agonist carbachol, opioids, and thyrotropin-releasing hormone (TRH) among others, have also been reported to induce the HTR.

Drugs such as the serotonin precursors tryptophan and 5-hydroxytryptophan (5-HTP), serotonin releasing agents (SRAs) like fenfluramine and ''para''-chloroamphetamine (PCA), and other agents like 1-methylpsilocin and 3,4-dimethoxyphenethylamine (DMPEA) stimulate serotonin receptors and can produce head twitches, but are not known to be hallucinogenic in humans. However, at least in the case of 5-HTP, this could be just be due to the very high doses required. It is notable in this regard that hallucinations are reported in a subset of cases of serotonin syndrome, although it is unclear at the present time whether these hallucinations are psychedelic in nature or are of a different etiology. While the SRA and mixed entactogen and psychedelic MDA likewise induces the HTR, findings are mixed and conflicting for the SRA and less hallucinogenic MDMA. The SRA dexfenfluramine produces wet dog shakes in rats, whereas the serotonin reuptake inhibitor fluoxetine has little or no effect on wet dog shakes. Amphetamine as well as ''para''-hydroxyamphetamine (given intracerebroventricularly) can also elicit the HTR at sufficiently high doses.

The preceding findings collectively suggest that while the HTR can be a useful indicator as to whether a compound is likely to display hallucinogenic activity in humans, the induction of the HTR does not necessarily mean that a compound will be hallucinogenic. In relation to this, caution should be exercised when interpreting such results.

Non-hallucinogenic serotonin 5-HT_2A receptor agonists

Some serotonin 5-HT_2A receptor agonists, including lisuride, 2-bromo-LSD (bromolysergide; BOL-148), ergotamine, 6-fluoro-DET, 6-MeO-DMT, Ariadne, zalsupindole (DLX-001; AAZ-A-154), ITI-1549, 25N-N1-Nap, and IHCH-7086 among others, are either non-hallucinogenic or are thought to be non-hallucinogenic in spite of activating the serotonin 5-HT_2A receptor. The HTR is among the only animal behavioral tests that can reliably distinguish between hallucinogenic and non-hallucinogenic serotonin 5-HT_2A receptor agonists. Although lisuride and other non-hallucinogenic serotonin 5-HT_2A receptor agonists do not produce the HTR in rodents, lisuride does produce the HTR in the least shrew, a non-rodent species that is said to be highly sensitive to serotonin 5-HT_2A receptor agonists. In any case, it is thought that partial agonism with sufficiently low efficacy for specific intracellular signaling pathways underlies the lack of HTR and psychedelic effects with non-hallucinogenic serotonin 5-HT_2A receptor agonists. However, other findings suggest that ergotamine may be non-hallucinogenic due to inability to efficiently cross the blood–brain barrier and peripheral selectivity, while lisuride and 2-bromo-LSD may actually be variably hallucinogenic at sufficiently high doses.

Serotonin administered by intracerebroventricular injection at high doses produces the HTR in animals. However, serotonin itself has been considered to be non-hallucinogenic in humans. This would be in accordance with the lack of inherent psychedelic effects with serotonin releasing agents, serotonin reuptake inhibitors, and serotonin precursors in humans. The HTR with high doses of serotonin in animals appears to be mediated by formation of more lipophilic ''N''-methylated psychedelic metabolites of serotonin, like bufotenin (''N'',''N''-dimethylserotonin).

Modulators of the head-twitch response

While the serotonin 5-HT_2A receptor mediates the HTR, other serotonin receptors, including the serotonin 5-HT_1A and 5-HT_2C receptors, appear to modulate the serotonin 5-HT_2A receptor-induced HTR. Serotonin 5-HT_1A receptor agonists like 8-OH-DPAT suppress the HTR, while serotonin 5-HT_1A receptor antagonists can augment it. In addition, LSM-775, which is a weakly hallucinogenic psychedelic in humans, does not induce the HTR in animals unless the serotonin 5-HT_1A receptor is blocked with WAY-100635, suggesting that serotonin 5-HT_1A receptor activation masks its psychedelic-like effects. The serotonin 5-HT_1A receptor agonist buspirone has been reported to suppress the hallucinogenic effects of serotonergic psychedelics in humans, while the serotonin 5-HT_1A receptor antagonist pindolol has been reported to markedly potentiate them. However, paradoxically, whereas the serotonin 5-HT_1A receptor full agonist 8-OH-DPAT suppresses the HTR induced by 5-hydroxytryptophan (5-HTP) or DOI, buspirone, a serotonin 5-HT_1A receptor partial agonist, has been shown to enhance the HTR induced by 5-HTP plus pargyline. The possible influence of serotonin 5-HT_2B receptor signaling on the HTR has been little-studied and is largely unknown.

Serotonin 5-HT_2C receptor agonists, for instance Ro 60-0175, CP-809,101, and ''meta''-chlorophenylpiperazine (mCPP), have been reported to suppress the HTR, while serotonin 5-HT_2C receptor antagonists, like SB-242084, have been reported to potentiate the HTR. However, in some studies, serotonin 5-HT_2C receptor inactivation, by antagonism with SB-242084 or SB-206553 or by receptor knockout, has been reported to diminish the HTR. The reasons for these contradictory findings are unclear. In any case, animal strain differences have been suggested. In addition, the influence of serotonin 5-HT_2C receptor signaling on the HTR may be bimodal, with a more recent study finding that the serotonin 5-HT_2C receptor antagonist RS-102221 enhanced the HTR at lower doses but inhibited it at higher doses.

A number of other drugs have also been found to modulate the HTR. Monoamine oxidase inhibitors (MAOIs) like harmine, iproniazid, pargyline, clorgyline, and tranylcypromine have been found to potentiate the HTR induced by serotonergic psychedelics and other serotonergic agents without inducing the HTR on their own. This is the case even with psychedelics that are not themselves monoamine oxidase (MAO) substrates, indicating that the potentiation is not simply due to inhibition of their metabolism. In contrast to MAOIs, serotonin reuptake inhibitors (SRIs), including citalopram, fluoxetine, fluvoxamine, and imipramine, do not affect the HTR induced by DOI. Conversely, serotonin transporter (SERT) knockout greatly reduces or even eliminates the psychedelic-induced HTR. This may be due to elevated serotonin levels and decreased serotonin 5-HT_2A receptor expression. Unlike SRIs, chronic administration of serotonin–norepinephrine reuptake inhibitors (SNRIs) has been found to decrease the DOI-induced HTR. The anticonvulsant phenytoin potentiates the HTR. NMDA receptor antagonists like phencyclidine (PCP), ketamine, and dizocilpine (MK-801) have been found to enhance the DOI-induced HTR as well.

A variety of other agents, including the β-adrenergic receptor agonist clenbuterol, AMPA receptor antagonists like tezampanel (LY-293558), metabotropic glutamate mGlu₂ and mGlu₃ receptor agonists like eglumegad and LY-379268, antipsychotics like haloperidol, antihistamines, μ-opioid receptor agonists like morphine, methadone, and pethidine, adenosine A₁ receptor agonists like ''N''⁶-cyclopentyladenosine, and the TAAR1 antagonist EPPTB, have been reported to inhibit the HTR induced by serotonergic psychedelics and/or other serotonergic agents in animals. Conversely, the metabotropic glutamate mGlu₂ and mGlu₃ receptor antagonist LY-341495 has been found to potentiate the psychedelic-induced HTR.

Serotonin depletion has been found to potentiate the HTR. This appears to be related to increased postsynaptic serotonin 5-HT₂ receptors.Heal, D. J., Metz, A., & Philpot, J. (1985). 5, 7-dihydroxytryptamine lesioning increases both 5-HT-2 mediated behavior and cortical 5-HT-2 receptor number in mice. Brit. J. Pharmacol., 85, 311P. https://scholar.google.com/scholar?cluster=6981396242978161484

History

The HTR was first described as an effect induced by LSD, independently by Winter and Flataker and by Keller and Umbreit, in 1956. Subsequently, it was described as an effect of large doses of 5-HTP, by Corne, Pickering, and Warner, in 1963. At first, the HTR was just a pharmacological curiosity and was not used as a tool in scientific research. In 1967 however, Corne and Pickering demonstrated the influence of a wide range of drugs on the HTR and proposed the HTR as a behavioral predictor of hallucinogenic effects in humans. The reliability of the HTR for identifying psychedelics is said to have been established by the mid-1970s. However, it has been said that the HTR test did not become widely used in studying serotonin 5-HT_2A receptor activation until the mid-2000s.

Studies published in the 1960s and 1970s had shown that serotonin receptor antagonists, such as cinanserin, methysergide, and cyproheptadine, blocked the hallucinogen-like effects of psychedelics in animals. Mediation of the HTR specifically via serotonin 5-HT₂ receptor agonism was first proposed by Peroutka, Lebovitz, and Snyder in 1981, followed by supporting studies by Ortmann and colleagues in 1982 and Leysen and colleagues also in 1982. Richard Glennon and colleagues further supported mediation of the hallucinogen-like effects of psychedelics by serotonin 5-HT₂ receptor agonism with subsequent studies, for instance employing drug discrimination, in 1983 and thereafter. However, the role of the serotonin 5-HT_2A receptor in the mediation of psychedelic-like effects, including the HTR, was not conclusively validated until studies with serotonin 5-HT_2A receptor knockout mice were published in 2003. It was found in 1985 that the non-hallucinogenic serotonin receptor agonist lisuride did not produce the HTR in animals and could antagonize the HTR induced by other drugs, leading to the suggestion that it was a low-efficacy partial agonist of the serotonin 5-HT₂ receptors.

Automation of the HTR assay was first described by Adam Halberstadt and colleagues in 2013. They developed a semi-automated assay using magnetometer-based detection. de la Fuente Revenga and colleagues developed a fully automated HTR test based on Halberstadt's work and published their system in 2019. Additional automated HTR systems, including ones employing deep learning techniques, were developed in 2020 and thereafter.

Other tests

The only other behavioral paradigms for assessment of psychedelic-like effects in animals at present are drug discrimination (DD) and, to a lesser extent, prepulse inhibition (PPI) and time perception. However, the HTR is far less costly and time-consuming than drug discrimination and hence has become much more popular in recent years. Other paradigms for assessing psychedelic-associated effects have also been studied but have not shown satisfactory consistency for general use. In the 2020s, a test of psychedelic-induced visual distortions in animals was published. This study marked the first evidence of psychedelic-induced visual distortions in animals. However, a much earlier test of psychedelic-related visual changes, described by the late 1960s, was a visual object size discrimination task in monkeys.Uyeno, E. T., Otis, L. S., & Mitoma, C. (1968). Behavioral evaluation of hallucinogenic trimethoxyamphetamines in squirrel monkeys (Saimiri sciureus). ''Communications in Behavioral Biology'', ''1''(1-A), 83–90. https://psycnet.apa.org/record/1969-12564-001

References

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External links

Psychedelic or Not: Of Mice, 5-HT2A Agonists, and Head Shakes (February 1, 2022) - Nate Seltenrich - Psychedelic Science Review (PSR)

Do Mice Hallucinate? Do Humans Head-Twitch? (February 24, 2023) - Psychedelic Alpha

LSD & The Head Twitch Response - Hamilton Morris (with Jason Wallach) - YouTube

Getting High On HIV Medication (short segment on head-twitch response) - Hamilton Morris - VICE - YouTube

The Search for Non-Hallucinogenic Psychedelics: Uncovering the Head Twitch Response - Mind & Matter Podcast

Head-twitch response (psilocybin) - Alex Kwan - YouTube

Animal testing techniques
Lysergic acid diethylamide
Medical terminology
Psychedelic drug research
Psychology experiments