The overall goal of this procedure is to obtain behavioral pre pulses inhibition data of the auditory startle response to identify the hearing threshold and a subjective tinnitus percept in rodents using a low cost set up. This is accomplished by first acquiring a full set of behavioral data and the healthy animal. Next hearing impairment is induced with a mild frequency specific acoustic trauma.
The third step is to obtain the same behavioral data in the hearing impaired animal. Then the data is analyzed to measure any hearing loss and a possible tinnitus percept. Ultimately, results can show not only hearing threshold changes, but also frequency specific tinnitus.
Percepts through the behavioral measurements of startled responses is Demonstrating the procedure will be consult in SA postdoc and Z ave, a PhD student in my laboratory. First, a PC with necessary equipment needs to be assembled. The total cost of this setup can be assembled for about 3, 500 euro compared to the standard equipment which can cost between 15, 000 and 20, 000 euro.
This includes a DA converter connected to a breakout box and an amplifier, an infrared webcam, and MATLAB software running an integrated development environment. The amplifier is connected to a speaker in a soundproof chamber, which is monitored by the webcam and is equipped with an insulated sensor board, a piazzo sensor, and a chamber to secure the rodent within the soundproof chamber. There is also a microphone for sound control.
Regardless of the quality of the amplifier and the speaker, the system must be calibrated with a measuring amplifier to determine the frequency transfer function for the system. Furthermore, the system must be tailored to the subject animal's hearing range. Take the animal from its home cage and put it headfirst into its holding tube.
Close the door, load the tube into the chamber on the foam feet, and the sensor switch off any ambient lights. Close the door to the chamber and wait 15 minutes to allow the animal to get accustomed to the setup. Start the program and define the parameters for stimulation.
Stimuli consists of pure tones with different frequencies. The test stimuli preceding the startle stimuli are presented at varying frequencies and intensities. Allow five minutes of recovery time between stimulus sets for a non-randomized approach, such as one test stimulus level fixed for all tested frequencies.
Before analyzing the data, remove invalid trials from the dataset such as trials where the animal moved before the startle stimulus. The validation of the single trials is very important as responses to the startle stimulus tend to be much stronger after the animal has moved. Such responses must be discarded.
Now, obtain the amplitude and latency of the startle response within a time window of the first 50 milliseconds After the startle stimulus, the response amplitude is the distance between the first maximum to the first minimum of the response. The latency is the time from stimulus start to response onset. The hearing threshold is obtained by fitting a boltman function to the complete response amplitude dataset of one frequency sorted for pre stimulus intensity of single trials.
The 50%point of the boltman function indicates the hearing threshold for this stimulation frequency. The responses to the pure startle stimulus without any pre stimulus can be used as reference for later calculations. To traumatize the rodents hearing, first anesthetize the animal inject three milliliters per kilogram of anesthesia subcutaneously, including a bolus of atropine.
Wait about five minutes and check its reflexes. To maintain anesthesia during measurements, continuously inject the same volume of anesthetic solution from a syringe pump. Over the course of every hour in the chamber, the animal should be kept warm by a heating pad.
For 75 minutes, subject the animal to loud, pure tone to induce an acoustic trauma. After waiting five days for the animal to stabilize, generate a new audiogram, a minimum of five days for tinnitus development is critical to obtaining reliable results. In the first subjective tinnitus paradigm, use the following parameters for stimulation.
Set the startle sound intensity to 105 decibels, SPL, and step the frequencies from one to 16 kilohertz in single oc. Begin by presenting a white noise background of 50 decibels, SPL, either with or without a 15 millisecond gap. If using a non-randomized approach, allow five minutes of recovery between the different stimulus sets.
Using different startle stimulus frequencies will give a rough estimate of perceived tinnitus frequency. On the second subjective tinnitus paradigm, use the following stimulation parameters, a startle sound intensity of 105 decibels, SPL with double click stimulus. Begin with a band pass filtered noise.
Background of 50 decibels, SPL. Again, if using a non-randomized approach, allow five minutes of recovery between the different stimulus sets. After removing any invalid trials from the dataset, perform the following calculations for each remaining trial.
First, over the first 50 milliseconds. After startle stimulus, measure the response amplitude and response latency. Second, normalize all of the data to a reference dataset.
The reference dataset is the response amplitude to the pure tone startle stimulus Without any pre stimulus, the test frequency is determined either by the pure tone or the center frequency of the band pass filtered noise. Calculate the mean response of each reference. Then normalize each response amplitude by dividing it through its reference.
Now, using the mean value of the normalized no gap condition, calculate the PPI of each tested condition. At this point, it is possible to calculate the PPI change after the trauma in percent for each tested frequency. Likewise, it is possible to directly compare the PPI before and after trauma.
Using the described paradigm, startle responses are easy to generate and analyze. The majority of the trials are valid. Invalid trials are easy to recognize.
They are marked by red squares and are omitted from the analysis. A typical behavioral threshold change acquired before and after an acoustic trauma at two kilohertz shows a clear hearing loss at two kilohertz. After inducing a hearing trauma, the normalized response amplitudes for stimulations, one octave below and one octave above the trauma were investigated below the trauma frequency.
No change of response pattern was found above the trauma frequency. The effect of the pre stimuli gap vanished indicating a misperception at this frequency. After watching this video, you should have a good understanding of how to obtain and process the behavioral data for hearing threshold and to need to percept evaluation in rodents with a low cost startle setup.
