Head Twitch Test

The test apparatus had eight individual observation chambers and was made of Plexiglas, measuring 10 cm × 10 cm × 12 cm per chamber, through which all animals were observed. An experienced blinded observer watched eight animals in tandem and recorded all head twitches on a multiple counter (Fisher Scientific, Inc., Pittsburgh, PA, United States). Mice were brought from the colony room in groups of eight, weighed, and allowed to acclimate to the test chamber for 15 min prior to testing. Head twitches were induced by DOI (3 mg/kg, i.p.), and mice were observed for 30 min beginning 5 min after dosing. The only exception to this was the initial DOI dose-response determination where the WT mice observation duration was 15 min instead of 30 min. {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268, {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495 and CBiPES were administered i.p., 30 min prior to DOI. When CBiPES was administered with {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495, CBiPES was dosed 5 min prior to {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495. Each experimental group consisted of eight animals per group except for the experiment testing DOI in mGlu₃ receptor KO animals (N = 10), the experiment testing the {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 dose-response relationship in WT mice (N = 6) and when {"type":"entrez-nucleotide","attrs":{"text":"LY341495","term_id":"1257705759","term_text":"LY341495"}}LY341495 was tested in mGlu₃ receptor KO animals (N = 16). The sample size used for these experiments were determined in part by availability transgenic mice. Separate groups of animals were used for each dose of DOI in all experiments with CD-1 (ICR) mice, and in all experiments utilizing KO/WT animals, mice were used at least twice, with a 7-day washout period between tests.

The methodology used for the DOI-induced HTR was fit for purpose as a relatively modest-high throughput behavioral assay for a range of drug discovery programs at the Lilly Research Laboratories. Statisticians and biologists worked in tandem to validate this in vivo assay (and other in vivo and in vitro assays). This validation included determining the number of animals required for individual behavioral experiments as well as the statistical tests used to evaluate whether a concentration- or dose-related effect was present. A critical part of the validation for the DOI-induced HTR including running these validating experiments with a trained observed (SFC), with substantial personal experience of measuring DOI-induced head twitches under these condition. On occasion, additional validating experiments were carried out such as an experiment where CBiPES statistically suppressed DOI-induced head twitches in a dose-related manner where a significant decrease in rat head twitches induced by DOI (3 mg/kg, i.p.) occurred for a 30 mg/kg CBiPES dose (approximately 10 head twitches with DOI alone down to about 4 head twitches with DOI/CBiPES). Furthermore, this mouse DOI-induced HTR assay was used for optimization of the physicochemical characteristics of mGlu₂ receptor PAM SAR that later resulted in mGlu₂ receptor PAMs that were both more potent and centrally penetrant. In a set of 12 compounds not described in this manuscript, the mean frequency of head twitches/30 min observation periods induced by DOI alone were 7.9 ± 1.2 (SD) in WT CD1 mice. The range of DOI-induced head twitches was (6.4–10.7). Only one experiment was the mean DOI-induce head twitch frequency lower than the 95% confidence intervals for the set of 12 experiments. Only for two experiments were the mean frequency of DOI-induced head twitches higher than the 95% confidence interval for the entire set of 12 experiments. MGlu₂ receptor PAMs significantly reduced the frequency of DOI-induced head twitches in doses ranging from 1 to 30 mg/kg. Additional internal converging validity of the DOI-induced head twitch assay for the mGlu₂ receptor PAM program was found in similar results for the same mGlu₂ receptor PAMs with respect to potency and maximal efficacy with hyperactivity induced by a NMDA receptor antagonist. Another internal converging validity of the DOI-induced head twitch assay for the mGlu₂ receptor PAM was found in similar results with respect to potency and maximal efficacy when testing for antidepressant-like activity in mice or rats on an operant differential-reinforcement-of-low rate (DRL) schedule of reinforcement. A range of other discovery projects (including selective mGlu₂ receptor orthosteric agonists, mGlu₅ receptor negative allosteric modulators, orexin receptor antagonists) also successfully utilized the DOI-induced head twitch assay either for supporting the development of new projects and/or utility as an early relatively modest-high behavioral throughput assay that was complimented by in vivo receptor occupancy (where available) and other physiological/behavioral assays.