A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.
In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in naïve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in naïve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).
1. Setup Assembling and Software Programming
2. Behavioral Determination of Hearing Thresholds (Audiograms)
3. Acoustic Trauma and Quantification of Hearing Impairment
4. Test for Acoustic Phantom Perception (Subjective tinnitus)
The startle responses of animals are easy to generate and to analyze. Figure 2B gives an overview of a typical result of one animal stimulated with a pure tone of 105 dB SPL without any prestimulus for 15 times. The majority of the trials are valid and invalid trials are easy to recognize (trials marked by red square). The response amplitudes and latencies are calculated only from valid trials.
A typical behavioral threshold change is given in Figure 3A. The audiogram of an exemplary animal acquired with the method described in 2 is given before (blue) and after (red) an acoustic trauma at 2 kHz (yellow area). A clear hearing loss is shown specifically at 2 kHz. The responses related to a subjective tinnitus percept can be seen in Figure 3B, the normalized response amplitudes of the same animal as above are shown exemplarily for stimulations one octave below and above the trauma as described in 4.5. The comparison of the responses to stimuli with and without gap before (blue) and after the trauma (red) allows an interpretation of a possible tinnitus percept. Below the trauma frequency no change of response pattern can be found while above the trauma the effect of the gap vanished after the trauma, indicating a mispercept at this frequency.
Figure 1. Flow diagram of the program used to acquire the behavioral thresholds and subjective tinnitus data. Note that this is only a simplified version of the program code. Abbreviations: GUI – graphical user interface; ISI – inter stimulus interval.
Figure 2. Auditory startle response (ASR) stimuli. A Schemes of the three different stimulation protocols used. Left panel: pre-pulse inhibition (PPI) of the ASR measured without any pure tone test stimulus before (green) the startle tone (red); the response period is depicted in blue. Center panel: gap / noise paradigm with pure tone startle stimulus presentation of different frequencies on a white noise background. Right panel: gap / noise paradigm with click startle stimulus presentation on bandpass filtered background of different center frequencies. B Exemplary auditory startle responses of 15 trials recorded with the threshold paradigm without any prestimulus at 1 kHz stimulation frequency. Three trials are counted invalid (red squares) as the animal moved already before the stimulation onset.
Figure 3. Exemplary results of ASR in one animal. A Behavioral threshold before (blue) and after (red) the acoustic trauma at 2 kHz (yellow area). The thresholds are calculated from the responses to the PPI modulated ASR protocol using the Boltzmann-function turning point as threshold value. Note that the hearing loss at 2 kHz amounts to more than 66%, while farer away from the trauma frequency one often can see even improvement of hearing thresholds. B Normalized response amplitudes (open circles: single trials, filled circles: means, whiskers: standard deviation) during stimulation with the gap / noise click ASR protocol (4.5) for 1 and 4 kHz center frequencies. Responses are sorted for trials without and with gap in the noise before and after the trauma at 2 kHz. Only at 4 kHz the effect of the gap vanishes after the trauma which indicates a subjective tinnitus percept around this frequency.
We present a cheap and easy to build setup for audiometric measurements in rodents based on pre-pulse inhibition of acoustic startle responses that can be used to determine behavioral hearing thresholds (= audiograms 10) and auditory phantom percepts like subjective tinnitus 11. Especially the latter measurements are in the focus of several recent reports 8,12,13,14 and can be seen as one prerequisite for electrophysiological investigations of the neuronal mechanisms underlying this disease. Using this method it is possible differentiate which animals did develop a subjective tinnitus percept after acoustic trauma and those that did not, and then further investigate these individuals, e.g., with electrophysiological recordings in primary auditory cortex.
A critical step in the analysis of the startle data after acoustic trauma is the normalization of the data to the startle amplitude that can maximally be elicited without preceding test stimulus: This is particularly important to distinguish reduced startle responses based on hearing loss from reduced PPI in tinnitus animals: The effects of the acoustic trauma are changing over time, as the animal partially recovers from it, but roughly 50% of the hearing loss is permanent. In contrast to reports mentioned above, where the auditory thresholds are tested but not used for calibration, we tried to minimize the effects of the different hearing thresholds of each frequency and the effect of the acoustic trauma itself by normalizing each response amplitude with a reference. Additionally we use two different kinds of protocols to assess any tinnitus percept, with the first (4.4) working better for animals tested over longer timescales from one week after the trauma on and the second “classical” (4.5) working better for animals tested within one week after the trauma.
A limitation of this method is clearly that one cannot assess the acute effects of an acoustic trauma. At least two days between the anesthesia and the first post-measurement should be chosen, as the animal has to recover from it. To obtain an estimate of acute hearing loss directly after trauma, brainstem audiometry (BERA) may be used.
The authors have nothing to disclose.
This work was supported by the Interdisciplinary Center for Clinical Research (IZKF, project E7) at the University Hospital of the University of Erlangen-Nuremberg.