Xenakis1 project protocol

Drug study: Haloperidol antipsychotic effects on behavioral phenotypes in 73 F1 hybrids of Collaborative Cross strains of mice (RIX)   (2020)

Xenakis JG, Giusti-Rodriguez P, Sullivan PF, Pardo-Manuel de Villena F, Zou F
With: Crowley JJ, Nonneman RJ, DeCristo DM, Ryan A, Quackenbush CR, Miller DR, Shaw GD, Zhabotynsky V

Project protocol - Contents

Workflow and sampling

Step Description Equipment Data collected
1 CC mice crossed to generate 73 RIX lines. - -
2 Baseline (pre-drug treatment) testing. At 8 weeks (-/+ 7 days) of age, body weight recorded. Additionally, extrapyramidal symptoms (EPS) assessed (-5 days) and mice subjected to open field test. Balance scale, inclined screen setup, open field arena Body weight, inclined screen test parameter (latency to move all four paws), open field test parameters (spontaneous locomotor activity, number of vertical beam breaks, time spent in center of arena, and number of beam breaks to determine stereotypic activity).
3 At 8 weeks of age (-/+ 7 days) or day 0, mice anesthetized and implanted with slow-release pellets containing either haloperidol or placebo. Two pellets implanted 2 days apart. Gas anesthetic delivery system, trocar -
4 24hr after the 2nd pellet implantation mice assessed for EPS (acute drug treatment) Inclined screen setup Latency to move all four paws.
5 At 28 days post-drug treatment mice weighed, and subjected to open field test in a similar manner as at baseline. Mice then immobilized and monitored to evaluate vacuous chewing movements (VCMs). Together these are considered chronic drug treatment assessment. Balance scale, open field arena, Observer XT video analysis system Body weight, open field test parameters (spontaneous locomotor activity, number of vertical beam breaks, time spent in center of arena, and number of beam breaks to determine stereotypic activity). Subtle and overt VCMs recorded as well as tongue protrusions and jaw tremors.
6 At +32 days post-treatment mice were sacrificed. Blood plasma collected from haloperidol treated mice. Mass spectrometer Plasma haloperidol concentration
Workflow Images:
Workflow summary


General Information for All Procedures: CC RIX mice were aged for approximately 8 weeks before being assessed for various phenotypes. Phenotype assessment occurred pre-drug (7 days before pellet implantation), after acute drug treatment (on day 4; one day after split pellet implantation), and chronic treatment with haloperidol or placebo (chronic drug: 28-35 days after pellet implantation). The incline screen test which assess extrapyramidal symptoms (EPS) was carried out both pre-drug, and after acute drug treatment. Open field test was carried out both pre-drug and after chronic drug treatment. Vacuous chewing movements (VCMs) were assessed after chronic treatment, as were haloperidol levels in blood plasma. Six mice were removed from the study due to technical difficulties in phenotyping.

Procedure 1: Body weight and haloperidol/placebo exposure

Equipment, software, and supplies

  • Balance scale
  • Trocar

Reagents and solutions

  • Haloperidol pellets (Innovative Research of America, Sarasota, FL, USA)
  • Isoflurane anesthesia


  1. Body weights of mice are recorded at the age of 7 weeks, and then at the end of the study approximately +32 days following haloperidol/placebo implantation.
  2. Mice are implanted with slow-release haloperidol pellets or placebo at approx 8 weeks (-/+ 7 days) and treated for approximately 30 days. Haloperidol pellets (3.0 mg/kg/day) are implanted subcutaneously with a trocar under 2 min of isoflurane anesthesia. Two pellets of incremental dosage are implanted 2 days apart. Placebo-treated mice are implanted with pellets containing the same matrix material but no drug.

Procedure 2: Incline screen test

Definitions & Abbreviations: EPS # Extrapyramidal symptoms

Equipment, software, and supplies

  • Wire mesh screen at 45 degree angle


  1. Mice are placed on a wire mesh screen inclined at 45 degrees and the time (latency) to move all four paws is recorded (up to a maximum of 60 s). EPS is assessed at 8 weeks (-/+7 days) (pre-drug treatment), and approximately 1 day after the second haloperidol/placebo is implanted (acute drug treatment).

Procedure 3: Open field test

Equipment, software, and supplies

  • Open field apparatus, arena with white Plexiglas floor and clear Pleixiglas walls that measures 40 x 40 x 30 cm (l x w x h); surrounded by infrared detection beams on the X, Y and Z axes (Accuscan Instruments, Superflex system, Columbus, OH, USA)
  • Test chamber, sound-attenuating box for the open field apparatus equipped with houselights and fans that measured 73.5 x 59 x 59 cm

Environmental Conditions

Testing Area

Acclimation Period: 1-2 hr

Light Source: Overhead fluorescent lighting

Light Intensity: Lux level 14

Temperature (°C): Not specified

Humidity: Not specified


  1. Mice are placed in one corner of the testing arena and allowed to explore the arena for a 30 min. Open field tests are carried out at 8 weeks (-/+7 days) (baseline or pre-drug treatment), and +28 days following pellet implantation (chronic drug treatment).

Procedure 4: Vacuous chewing movements assessment

Definitions & Abbreviations: VCMs # Vacuous chewing movements

Equipment, software, and supplies

  • Plastic collar
  • JVC Everio digital camcorder
  • The Observer XT (Noldus Information Technology, Wageningen, The Netherlands)

Reagents and solutions

  • Isoflurane anesthesia


  1. Mice are briefly anesthetized with isoflurane and restrained with a plastic collar for 25 min. The collar partially immobilizes the neck but still allows head movements to allow for video recordings of jaw movements. The first 10 min of video are not analyzed in order to allow the subject to acclimatize to the head collar. The last 15 min of the video are scored for orofacial movements, specifically tongue protrusions, jaw tremors, overt chewing movements and subtle chewing movements. The videos are scored using the Observer XT observational data analysis program.
  2. Videos are randomized and scored by a single-blinded rater to increase consistency and to reduce any deviation or bias between raters. Individual events of each movement are counted. Subtle chewing movements are defined as instances of vertical jaw movement in which the inside cavity of the mouth cannot be seen and the jaw is not open for a long period of time. Overt chewing movements occur when larger vertical movement are observed in which the cavity can be seen and the jaw is open for an extended period of time.

Procedure 5: Haloperidol concentration in blood plasma

Equipment, software, and supplies

  • Mass spectrometer


  1. On day 35 post-drug treatment mice are sacrificed by cervical dislocation. Blood plasma is collected from haloperidol-treated mice, centrifuged to isolate the plasma, and the drug concentration assessed by mass spectrometry.


Primary References

Giusti-Rodríguez P, Xenakis JG, Crowley JJ, Nonneman RJ, DeCristo DM, Ryan A, Quackenbush CR, Miller DR, Shaw GD, Zhabotynsky V, Sullivan PF, Manuel de Villena FP, Zou F. Antipsychotic Behavioral Phenotypes in the Mouse Collaborative Cross Recombinant Inbred Inter-Crosses (RIX). G3 (Bethesda). 2020 Sep 2;10(9):3165-3177. doi: 10.1534/g3.120.400975.   PubMed 32694196     FullText