Head Twitch Response (HTR) Test for 5-HT2A Receptor Activity

The head-twitch response in rodents indicates stimulation of the 5-HT2A receptor. This knowledge helps researchers design experiments to understand how psychedelics work.

March 01, 2019 -

The Head Twitch Response (HTR, also called “wet-dog shakes”) is a widely used behavioral assay in rats and mice to test for activation of the serotonin 5-HT2A receptor. In 1967, Corne and Pickering first recognized HTR as a possible predictor for hallucinogenic effects in humans.1 The 5-HT2A receptor and other 5-HT subtypes were unknown at the time of their work.

Building on the Work of Corne and Pickering

In 1982, researchers observed a relationship between the 5-HT2 receptor and the HTR produced from hallucinogens.2 In this work, there was a significant correlation between 19 mescaline antagonists and their affinity for the 5-HT2 receptor. Further, a study in 1990 found that ketanserin blocks the HTR to DOI (1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane).3 Also, Schreiber et al. showed in 1995 that the activation of 5-HT2A is responsible for HTR and the dopamine receptors D1 and D2 and 5-HT1A play modulating roles in HTR expression.4

In 2007, researchers used 5-HT2A knockout mice to study the HTR.5 This study found that several substances including psilocybin, mescaline, and LSD did not induce the HTR in these genetically engineered creatures. Put another way, the study found that 5-HT2A is responsible for HTR by using mice that did not have the 5-HT2A receptor.

Designing Experiments to Test Receptor Function

Scientific studies dealing with receptor function can be difficult to understand. These studies often use compounds that block receptors as well as drugs and other chemicals that may partially block the receptor, make it more sensitive, or modify its function in some other way.  When designing a study, researchers break it down step by step using the scientific method and posing questions they want to answer to test their hypothesis. The steps through an experiment using HTR to examine the effects of an experimental drug on the 5-HT2A receptor may go something like this:

  1. Administer the known 5-HT2A full agonist, 5-hydroxytryptophan (5-HTP) to mice. This should elicit the HTR.
  2. Compare the results of 1 above with the HTR after administering an experimental drug along with 5-HTP.
  3. If the HTR is reduced or stops, this means the experimental drug is a 5-HT2A antagonist. If the HTR increases, this means the experimental drug is a 5-HT2A agonist. However, the experimental drug could also be acting as an allosteric modulator of the 5-HT2A receptor and not acting on the receptor itself. Further testing using functional assays is required to determine if this is the case.
  4. Administer the experimental drug along with a known substance that blocks (antagonist) 5-HT2A (like ketanserin, for example, or use knockout mice as described previously). The antagonist makes the receptor unable to respond. Under these conditions, HTR should not occur. This step verifies that the experimental drug is working via stimulation of the 5-HT2A receptor.

An interesting (and not too complicated) paper to start with is a 2015 study by Zhuk et al., “Research on acute toxicity and the behavioral effects of methanolic extract from psilocybin mushrooms and psilocin in mice.” 6 The researchers used 5-HTP and HTR to test if there was a difference between the effects of mushroom extracts (containing psilocin along with other compounds) and pure psilocin on mice. A PDF copy of the full study is available here.

  1. Corne SJ, Pickering RW. A possible correlation between drug-induced hallucinations in man and a behavioural response in mice. Psychopharmacologia. 1967;11(1):65-78. doi:10.1007/BF00401509
  2. Leysen JE, Niemegeers CJ, Nueten JMV, Laduron PM. [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution, and functional role. Mol Pharmacol. 1982;21(2):301-314. http://molpharm.aspetjournals.org/content/21/2/301.
  3. Nissar A. D, Billy R. M, Richard A. G. Withdrawal from chronic treatment with (±)-DOI causes super-sensitivity to 5-HT2 receptor-induced head-twitch behaviour in mice. European Journal of Pharmacology. 1990;186(1):115-118. doi:10.1016/0014-2999(90)94066-7
  4. Schreiber R, Brocco M, Audinot V, Gobert A, Veiga S, Millan MJ. (1-(2,5-dimethoxy-4 iodophenyl)-2-aminopropane)-induced head-twitches in the rat are mediated by 5-hydroxytryptamine (5-HT) 2A receptors: modulation by novel 5-HT2A/2C antagonists, D1 antagonists and 5-HT1A agonists. J Pharmacol Exp Ther. 1995;273(1):101-112. http://jpet.aspetjournals.org/content/273/1/101.
  5. González-Maeso J, Weisstaub NV, Zhou M, et al. Hallucinogens Recruit Specific Cortical 5-HT2A Receptor-Mediated Signaling Pathways to Affect Behavior. Neuron. 2007;53(3):439-452. doi:10.1016/j.neuron.2007.01.008
  6. Zhuk O, Jasicka-Misiak I, Poliwoda A, et al. Research on Acute Toxicity and the Behavioral Effects of Methanolic Extract from Psilocybin Mushrooms and Psilocin in Mice. Toxins. 2015;7(4):1018-1029. doi:10.3390/toxins7041018