Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior

The behavioral assessment of experimental pain is essential for the analysis of pain mechanisms and the validation of therapeutic targets. Arthritic pain, in particular, is significantly associated with negative affective states and disorders. Here we present a standardized method for the quantitative analysis of audible and ultrasonic (25 ± 4 kHz) vocalizations in awake rats as a measure of higher integrated behavior in a model of arthritic pain. A bat detector and a condenser microphone were used to record ultrasonic and audible vocalizations, respectively, in response to innocuous and noxious mechanical stimulation of the knee before and after induction of acute arthritis in one knee. A computerized system was used to analyze number and duration of the filtered signals. For the behavioral tests, the animal was placed in a customized recording chamber to ensure consistent stimulus application and stable recordings and to eliminate any movement-induced noise. Noxious stimuli produced stronger vocalizations than innocuous stimuli. Both audible and ultrasonic vocalizations to innocuous (allodynia) and noxious (hyperalgesia) stimuli increased after the induction of acute arthritis. These changes were accompanied by increased knee joint circumference, lowered hind limb withdrawal thresholds and reduced exploratory behavior in the same animals. The computerized analysis of audible and ultrasonic vocalizations is a valid, quantitative, reliable and convenient method to measure pain-related behavior.