1College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin, 2The Waggoner Center of Addiction and Alcohol Research, University of Texas at Austin, 3Department of Psychology, University of Michigan, 4Institute for Neuroscience, University of Texas at Austin, 5Department of Psychology, University of Texas at Austin
Maier, E. Y., Ma, S. T., Ahrens, A., Schallert, T. J., Duvauchelle, C. L. Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats. J. Vis. Exp. (41), e2041, doi:10.3791/2041 (2010).
*= important points
Figure 1: Spectogram display of a 22-kHz range USV. Long calls in the 22-kHz range are elicited in conjunction with aversive stimuli and are thought to reflect a negative emotional state.
Figure 2: Spectogram display of 50-kHz USVs. Short "flat" and "frequency modulated" 50-kHz USVs are elicited in anticipation and during cocaine self-administration. They are associated with positive affect.
Figure 3: Cocaine-Induced 50-kHz range USVs. Graph depicts USVs (10-sec bins) elicited before and after self-administered cocaine injections (0.75 mg/kg/inj) in a representative rat on Day 5 of cocaine self-administration sessions. Immediately after placement within the operant chamber and prior to cocaine availability, cocaine-anticipatory USVs were elicited and during self-administration, cocaine-induced USVs were emitted.
Rats vocalize in ultrasonic frequencies that serve a social and communicatory function and express subjective emotional states 1,2. 22-kHz USVs are triggered by events that cause anxiety and distress, such as footshock cues, the presence of predators, aversive drugs and social defeat 2,3. Therefore, these typically long USVs (>0.3s) are thought to represent a state of negative affect of the animal. Conversely, shorter 50-kHz USVs (>0.3s) are emitted during and in anticipation of positive encounters, such as feeding, mating and social play, and have thus been linked to a positive emotional state of the animal 2,4. These calls have also been associated with reinforcing events related to the activation of the dopaminergic system 5. For example, increased 50-kHz USVs have been elicited by systemic injections of cocaine 6 or d-amphetamine 7 and environments previously paired with drugs of abuse 8-10.
It has been proposed that increases in locomotor activity in rodents relate to enhanced motivation and appetitive behavior 11-14. However, this relationship is indirect as best. By studying the emergence and persistence of USVs of particular frequencies, it is possible to gain insight to emotional effects of various aspects of drug experience.
In this experimental setup, we collected USV recordings immediately before and during drug self-administration sessions. This allowed us to examine the development of drug expectation during the non-drug interval just prior to the start of the session and to assess the direct effects of cocaine on USVs. The drug self-administration method allows rats to directly control their own drug intake levels animals and develop accurate expectations of the timing and frequency of drug administration. As a result, USVs detected during these sessions reflect accurate expectations of drug delivery. In addition, USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs), in rats receiving experimenter-administered drugs, and during drug extinction trials. Therefore, changes in affect associated with drug anticipation, non-contingent drug administration and drug abstinence can also be determined. Analyses of USV changes over the course of short- and long-term drug exposure could provide a more detailed interpretation of drug exposure effects on affective functioning.
In addition to drug self-administration experiments, USV recordings in operant chambers can also be used for a variety of other behavioral manipulations associated with reward or aversion, such as alcohol drinking, food reinforcement, and fear conditioning and extinction procedures.
Important Methodological Details
Since the MedPC software can be used to precisely manipulate experimental conditions, such as lever availability and lighting conditions, its use in combination with USV recording and analyses allows emotional effects of drug-associated cue presentations (i.e., light presentations or other sensory stimuli) and the direct effects of drug intake to be determined. Therefore, to ensure overlapping timelines of self-administration procedures and USV recordings, it is crucial that both software programs (e.g., MedPC and RECORDER) are activated simultaneously at the start of each session.
Due to the enormous amount of digital space required to store USV data (e.g., a 10-minute sound file requires approx 250 MB), caution must be taken when multiple animals are run on a single computer system at the same time. To avoid recording errors, the RAM size of the recording computer system needs be substantial (1-2 GB). Other ways to decrease recording errors include lowering the USV resolution during recording and/or reduce the number of animals run at one time. In addition, it is important that the researcher is vigilant to all aspects of data collection throughout the experimental procedure. For instance, the accumulation of a large amount of recording data on the hard drive may cause the USV recording program to pause. In this case, the recording program would need to be restarted immediately to avoid loss of USV data and de-synchronization of USVs with other behavioral measures.
Environmental conditions may influence USV calling. For example, the touch by an unfamiliar human can cause the animal to emit negative 22-kHz calls, which could be mistaken as an aversion to the experimental conditions. Therefore, intensive handling prior to experimental manipulations is an important aspect of behavioral experiments. In addition, since novel environments can also elicit non-specific USV calls, animals should be habituated to the test environment if possible.
No conflicts of interest declared.
This work has been funded by NIH Grants RO1DA014640 and 3R01DA014640-05S1, The University of Texas VP Research Office (C.L.D), NIDA Drug Supply Program, and the University of Texas Waggoner Center for Alcohol and Addiction Research Bruce-Jones Graduate Fellowship (E.Y.M). We thank Leah McAleer, Mohamed Abdalla, Neha Thakore, Byron Barksdale, Tiffany Nguyen, Tian Tian, Hunter Owen, Proy Phongsawad, Helen Reed, Rachel Chavana, Linda Ju and Rosie Maddox for their assistance in USV data analyses.
|Operant Conditioning Behavior (Drug Self-administration) Test Package for Rat||Med Associates, Inc.||MED-008-CT-B1|
|Infrared Source and Detector (Photobeams)||Med Associates, Inc.||ENV-253SD ENV-253|
|Med PC Software||Med Associates, Inc.||SOF-735|
|Single speed syringe pump||Razel Scientific Instruments||Model R-E|
|45 mg sucrose pellets||Bio-Serv||F0042|
|Catheter cannula||Plastics One||C313G-5UP|
|Avisoft-UltraSoundGate 416H||Avisoft BioAcoustics||Part #34163|
|Ultrasonic microphones||Avisoft BioAcoustics||Part #40011|
|Avisoft-RECORDER Software||Avisoft BioAcoustics||Part #10302|
|Avisoft-SASLab Software||Avisoft BioAcoustics||Part #10101|
|PC Computer for USV recording||Dell|
|PC Computer for USV data analyses||Dell|