Models of Anxiety: Ultrasonic Vocalizations of Isolated Rat Pups

Lucianne Groenink1, P. Monika Verdouw1, Ruud van Oorschot1, Berend Olivier2

1 Psychopharmacology, Department of Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht, The Netherlands, 2 Deptartment of Psychiatry, Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.18
DOI:  10.1002/0471141755.ph0518s43
Online Posting Date:  December, 2008
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Described in this unit is the ultrasonic distress vocalization test in rat pups. This test is a reliable method for detecting anxiolytic properties of test compounds. In this test, ultrasonic vocalizations (30 to 50 kHz) are elicited by separating rat pups of 9 to 11 days of age from their mother and littermates for a brief period of time. The test can be performed under two different stress conditions. Pups are placed in isolation in either a warm (37°C) or cold (18°C) environment for 5 min. The total number and duration of ultrasonic calls emitted by the pups during this period is used as an index of anxiety. Established anxiolytic compounds, including benzodiazepines, serotonin1A receptor agonists, and selective serotonin reuptake inhibitors (SSRIs), consistently reduce the number and cumulative duration of these ultrasonic distress vocalizations. Curr. Protoc. Pharmacol. 43:5.18.1‐5.18.14. © 2008 by John Wiley & Sons, Inc.

Keywords: anxiety; ultrasonic vocalization; distress; rat pup; negative geotaxis

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Young adult male and female Wistar rats, age 10 to 12 weeks, for breeding program, or pregnant dams, gestation day 9 (e.g., Harlan/CPB Zeist)
  • Vehicle for dissolving test compound
  • Test compound
  • Silicon oil
  • 100 × 50 × 60–cm and 425 × 266 × 180–mm cages
  • Custom made ultrasonic vocalization testing cylinder: Plexiglas cylinder (19‐cm i.d., 15‐cm height) with a lid through which a microphone can be inserted (Fig. )
  • Audio apparatus—complete system consists of:
    • Microphones, or mini‐3‐bat detector (Ultra Sound Advice;
    • Control unit, including amplifier, audio filters, and software for data acquisition and processing, such as Avisoft‐SASLabPro (Avisoft Bioacoustics;, Sonotrack (Metris B.V.;, ultrasonic vocalization detector (Med Associates), or UltraVox (Noldus Information Technology)
    • Alternatively, a system can be built with separate components, including ultrasonic microphone with amplifier, audiofilters, analog‐to‐digital converter, oscilloscope, and customized signal‐detection software (see, e.g., Fish et al., ; Satow et al., )
  • Computer for data recording and statistical analysis software: e.g., SPSS or SAS (
  • Cold and hot plates: 20‐cm stainless steel plates held at either 18° or 37°C by circulating thermostatted water
  • Refrigerated/heating circulators (e.g., Julabo F12‐ED) to either cool (18°C) or warm water (37°C) and to circulate the thermostatted water through the steel plates
  • Separate warm plate for keeping nest at 37°C
  • Animal balance, accuracy 0.1 g (e.g., Mettler, type PG5000)
  • 1‐ml syringes
  • 25‐G × 5/8‐in. needles
  • Waterproof markers in various colors
  • Inclined (20°) screen (Fig. )
  • Digital thermometer with thermistor probe (e.g., Keithley, 871A) for rectal temperature measurement
  • Stopwatch
NOTE: Most USV studies have been performed with pups from an in‐house breeding program. This is not absolutely necessary, as it is also possible to order pregnant dams (e.g., Hodgson et al., ). However, prenatal stress could result in behavioral and neurochemical alterations in the offspring (Weinstock, ) and might affect test outcome.
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Literature Cited

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Key References
   Gardner, 1985b. See above.
  Description of a simple screening method to measure the effects of anxiolytic drugs on USVs in rat pups.
   Insel and Winslow, 1991. See above.
  Background data about rat pup USV and some psychopharmacology of USVs.
   Olivier et al., 1994. See above.
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