# Head-twitch 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 when the serotonin 5-HT$_{2A}$ receptor is activated. 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 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. Other paradigms have also been studied but have not shown satisfactory consistency.

## 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 10 milliseconds and each HTR consists of 5 to 11 individual 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 68 head 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.
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.

## 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 0.1 mg/kg DOI, maximal HTR is observed at 1 to 10 mg/kg, and lesser HTR is observed at 3 to 20 mg/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 (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 mGlu$_2$ 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.

## 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, ALD-52, 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-hallucinogens 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-HT$_2$ receptors. Examples of these agents include NMDA receptor antagonists like phencyclidine (PCP), certain benzodiazepines and Z-drugs like estazolam, triazolam, and zopiclone, α$_2$-adrenergic receptor antagonists like yohimbine, muscarinic acetylcholine receptor antagonists like atropine and scopolamine, serotonin 5-HT$_{1A}$ receptor antagonists like WAY-100635 and UH-301, and CB$_1$ receptor antagonists like rimonabant. 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 that hallucinations are reported in a subset of cases of serotonin syndrome. 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.
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.
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 mGlu$_2$ and mGlu$_3$ receptor agonists like eglumegad and LY-379268, antipsychotics like haloperidol, antihistamines, μ-opioid receptor agonists like morphine, methadone, and pethidine, adenosine A$_1$ 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 mGlu$_2$ and mGlu$_3$ 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-HT$_2$ receptors.

## History

Drugs inducing HTR from Corne & Pickering (1967) :
| Drug | ED$_{50}$Tooltip Median effective dose (mg/kg s.c.Tooltip subcutaneous injection) | Time to max effect (min) | Drug class |
| LSDTooltip Lysergic acid diethylamide | 0.045 | 9–11 | Psychedelic |
| MLD-41Tooltip N1-Methyl-lysergic acid diethylamide | 0.074 | 4–6 | Psychedelic |
| Ergometrine | 10 | 14–16 | Psychedelic |
| Psilocin | 1.3 | 4–6 | Psychedelic |
| Psilocybin | 1.1 | 4–6 | Psychedelic |
| DMTTooltip Dimethyltryptamine | 2.8 | 2–4 | Psychedelic |
| Bufotenin | 15 | 14–16 | Psychedelic |
| AMTTooltip α-Methyltryptamine | 7.9 | 44–46 | Psychedelic |
| 5-HTPTooltip 5-Hydroxytryptophan | 75 | 9–11 | Serotonin precursor |
| Mescaline | 9.6 | 19–21 | Psychedelic |
| TMATooltip 3,4,5-Trimethoxyamphetamine | 11 | 14–16 | Psychedelic |
| α-Ethylmescaline | ~20 | 14–16 | Psychedelic |
| DMPEATooltip 3,4-Dimethoxyphenylethylamine | 15 | 9–31 | Psychedelic |
| Scopolamine | 4.2 | 9–11 | Antimuscarinic |
| Atropine | 8.9 | 2–16 | Antimuscarinic |
| Trihexyphenidyl | 7.6 | 14–16 | Antimuscarinic |
| Ditran | 11 | 4–6 | Antimuscarinic |
| Phencyclidine | 0.36 | 4–21 | NMDAR antagonist |
| Yohimbine | 8.4 | 14–31 | α$_2$R antagonist |
| Notes: Drugs that did not produce the HTR in the study included ALD-52, BOL-148, ergotamine, methysergide, αETTooltip α-ethyltryptamine, serotonin, nalorphine, atropine methylnitrate, amphetamine, epinephrine, α-propylmescaline, α-butylmescaline, pheniprazine, tranylcypromine, acetylcholine, histamine, bradykinin, and hypertonic saline. ||||
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-HT$_2$ 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-HT$_2$ 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-HT$_2$ 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.