This video demonstrates a procedure for assessing the effects of cocaine on ultrasonic vocalizations or USBs. In rats during drug self-administration, trained rats are placed into a self-administration chamber where they press a lever to receive a cocaine reward and USBs are recorded in real time. The created sound file is converted into a spectogram, which allows the recorded USBs to be visible and audible to human experimenters.
The rat USBs are then differentiated from background noise and individual parameters of confirmed USBs are exported to an Excel file. This way USBs can be used as real-time measures of the rat's emotional response during drug self-administration. Hi, I'm Esta Meyer from the laboratory of Dr.Christine Hel in the College of Pharmacy pharmacology or toxicology division at the University of Texas that Austin.
Today we'll show you procedure for assessing rat ultrasonic vocalizations during drug self-administration. We use this procedure in laboratory to study the effects of cocaine on rat vocalizations. So let's get started.
The first preparatory step in performing combined drug self-administration and ultrasonic vocalization or USV experiments is to train rats to press a lever for a food reward. Once trained rats undergo a jugular catheterization surgical procedure to allow for drug self-administration. The apparatus used for drug self-administration consists of a single lever operant chamber, equipped with house and stimulus lights and three sets of photo beams.
The operant chamber is housed within a larger sound attenuating chamber to limit outside noise and light to detect and record USPS ultrasonic microphones are securely mounted in the center of the operant chambers. Microphones are connected to data acquisition hardware located outside of the operant chamber, and recordings are collected on a PC computer system using recorder, multi-channel recording software. The animal's behavioral responses are recorded using a Med Pentium computer equipped with med PC software, the med PC programs, and the AVA soft recorder must be loaded and ready before the animals are placed into the operant chambers.
In the Med PC menu, load the experimental procedure for the rats for their assigned operant chambers to start the program. Simultaneously with the USV recording, the stark command must be ready to be issued. Now the microphones must match with the rats in their assigned operant chambers on the microphone recording computer under the configuration settings of the AVA soft recorder software labeled the channel according to the animals, set the links of the recording intervals and define the keyboard key to start or stop the recording for the individual channel.
With everything set up, let's see how to conduct a recording session. To begin a recording session, gently remove the animal from its home cage and flush the indwelling intravenous catheter with 0.05 milliliters of a heparinized saline solution to detect possible catheter malfunctions. Next, connect the animal to a swivel mounted on the operant chamber that allows either drug or vehicle administration during the self-administration sessions.
Now gently place the animal into the opera and chamber and quietly close the doors. Once the doors are closed, begin recording. Because the drug self-administration program and the USB recording software are on two separate computer systems, they need to be started simultaneously.
If computer systems are closed together, this can be performed by one experimenter. The individual USPS are recorded and timestamped continuously throughout the session. The Med PC program controlling and recording events within the operant chamber can be written to any specification for the first 10 minutes of each session.
Rats occupy a darkened chamber without access to the lever. After exactly 10 minutes, the house light is illuminated and the lever is extended into the chamber indicating cocaine or vehicle availability. After each lever response, the lever retracts while the drug or vehicle is delivered and the stimulus light above the lever illuminates with the duration of the drug infusion.
After 20 seconds, the lever extends back into the chamber signaling drug availability. The session continues in this manner for the desired session duration. For this experiment, drug self-administration was programmed to be available for 60 minutes.
At the end of the session, the lights within the chamber turn off. The lever is retracted, and drug is no longer available at the end of the drug self-administration session. The USB recording program must be manually terminated on the USB recording dedicated computer system.
After the drug self-administration and USB recording sessions are terminated, gently remove the animal from the operant chamber, disconnect it from the swivel and flush the endowing catheter with 0.05 milliliters of heparinized saline to clear any drug residues from the catheter. The next step is to begin analyzing the data. USB analysis must be performed carefully to ensure accuracy.
SaaS lab pro analyzing software converts the dot wave file into a spectogram that allows the recorded US VS.To be visible and audible for the human experimenters. The resulting spectogram can be modified for individual preferences using the available configuration settings. Recorded sounds are displayed in the range of zero to 100 kilohertz including rat US vs.
And background noise generated by the fan in the sound attenuating chamber. To filter out this background noise, set the high pass of the spectogram to 10 kilohertz or higher as most of it lies in the range from zero to 10 kilohertz now enable automatic measurements and all sounds above. The cutoff frequency will be automatically detected by the AVA Soft software.
Using the tool remove gaps between detected elements from sound file. All parts of the original dot way file without sound can be erased. The shortened dot way file can now be saved and a shorter newly created spectogram can be used for a faster analysis.
Since both background noise and rat USBs are detected by the software, it's necessary to listen to the detected sounds during assessment. To verify and differentiate rat USPS from background noise, the identified background noise must be manually deleted from the spectogram. The automatic parameter measurements window offers a selection of measurements.
For example, time duration, bandwidth, frequency and amplitude. For the individual USV that can be transferred to an Excel file after the analysis to export only the confirmed rat USBs into an Excel file, the list of detected sounds created by the automatic parameter measurements window must be updated. After the assessment, the created list can be exported to an Excel file under the tool export by selecting copy parameter measurement headings and copy parameter measurement values for entire file.
The created Excel file contains information about the properties of the USBs and most importantly for this experiment, the exact times of the emitted USBs. This allows correlation of USBs to the exact time before and after the drug is self-administered. This way USBs can be used as real-time measurements of the rat's emotional response at specific instances during the experimental procedure.
Shown here is a spectogram of a 22 kilohertz USV. Long 22 kilohertz calls are elicited in conjunction with averse of stimuli and are thought to reflect a negative emotional state. This spectogram displays 50 kilohertz USBs of short, flat, and frequency modulated USBs elicited in anticipation and during cocaine self-administration.
They're associated with positive effect. Shown here are real-time 50 kilohertz USBs. The graph depicts USBs elicited before and after self-administer cocaine injections in a representative rat.
On day five of cocaine self-administration sessions Prior to cocaine availability, cocaine, anticipatory USBs were elicited. And during self-administration, cocaine induced USBs were emitted. We've just shown you how to record and assess red US vs.
Elicited during cocaine self-administration sessions. When doing this procedure, it is important to remember to set up the programs correctly so they can be started simultaneously and run without errors. So that's it.
Thanks for watching and good luck with your experiments.
