Name: pupillometry to assess auditory sensation in guinea pigs
Uploaded: 2023-01-06T13:25:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Pupillometry to Assess Auditory Sensation in Guinea Pigs at JoVE.com

This work was supported by the NIH (R01DC017141), the Pennsylvania Lions Hearing Research Foundation, and funds from the Departments of Otolaryngology and Neurobiology, University of Pittsburgh.

The authors have no conflicts of interest to disclose.

This protocol demonstrates the use of pupillometry as a non-invasive and reliable method to estimate auditory thresholds in passively listening animals. Following the protocol described here, call-in-noise categorization thresholds in normal hearing GPs were estimated. Thresholds estimated using pupillometry were found to be consistent with those obtained using operant training62. Compared to operant training, however, the pupillometry protocol was relatively straightforward and quick to set up and acquire data. Each data acquisition session (per SNR level) lasted about 12 min, which resulted in 1-2 h of experimental sessions (across SNR levels) per animal per day49. Data acquisition could be completed in about 7-10 days (depending on the number of SNR levels used). Although the oddball paradigm was used for call-in-noise categorization threshold estimation in this manuscript, this pupillometry protocol can be adapted to easier versions of oddball paradigms, where just one call exemplar is used, or to other stimulus paradigms using a wide range of complex or simple stimuli49.
The method is not without disadvantages. First, the current protocol requires the implant of a head post to fix the head during these experiments. Head post implant surgery and recovery would add a minimum of 2 weeks to the timeline of the experimental protocol. It is possible that this step can be avoided by using other methods of non-invasively immobilizing awake animals during experiments-for example, by using custom 3D-printed helmets63 or deformable thermoplastics64. Further experiments are necessary to explore these solutions. Second, animals could rapidly habituate to deviant stimuli as well, resulting in decreasing pupil dilation responses over the course of an experimental session. This effect could be minimized by restricting experimental sessions to short durations (~12 min) and presenting only a limited number (8) of deviant stimuli. Furthermore, an air puff delivered after the deviant stimuli can ensure that animals remain engaged with the auditory stimuli. Third, because of this rapid habituation, several days may be required to complete data acquisition. By only testing SNR values that densely sample the steepest parts of the psychometric curve, the total number of experimental days can be minimized. Fourth, animals may not keep still during experiments, or blink excessively or close their eyes during experiments. These factors are a function of species and acclimation and show a high degree of individual variability. GPs are naturally docile, and by acclimating them well to the experimental setup, motion and blink artifacts can be minimized. Spontaneous blinks and saccades are typically quite rare in guinea pigs49, but this could be a function of the species as well. Finally, as mentioned earlier, pupil dynamics in humans have been associated with a number of neuropsychiatric disorders. While the experimental animals used here are assumed to be neurotypical, this caveat must be kept in mind while interpreting results.
While one hardware implementation of pupillometry is described here (using a commercially available eye tracker and neural data acquisition system), the equipment required is expensive and not economical to scale-up. However, other custom solutions based on the same underlying principle of infrared-based eye tracking that are more cost-effective, are available. For example, one study used custom components and custom video processing algorithms to extract pupil diameter from the recorded video22,25. Recently developed deep-learning algorithms are also able to extract pupil diameter from videographic data65,66. These solutions could more than halve the cost of pupillometry rigs. The trade-off here is between expense and time-while commercial solutions are more expensive, they are turn-key solutions that can be used out of the box. On the other hand, custom solutions are cost-effective and scalable, but require expertise to set-up, and the time needed to develop custom analysis pipelines.
Although the protocol detailed here was performed in normal hearing GPs, pupillometry could be relatively easy to use in other animal models of hearing impairment with appropriate changes to stimulus type and parameters. This would allow for characterizing the effects of hearing loss across a range of stimulus types and species, which could potentially yield novel observations. Since pupillometry is a non-invasive technique that has also been used extensively in humans, by using the same stimuli used for animal subjects, pupillometry can be used to compare the effects of various auditory pathologies across species. For example, a recent meta-analysis in humans showed that speech-in-noise perception deficits arising from moderate noise exposure were best observed when complex and temporally varying stimuli were used67. The estimation of call-in-noise categorization thresholds by pupillometry demonstrated here could be used as one such task using complex stimuli to evaluate the effects of noise exposure in GPs. The assessment of hearing at a behavioral level using these methods would complement electrophysiological and anatomical methods and could be part of the standard toolkit for evaluating various known hearing disorders.
In conclusion, the following points are critical for successful acquisition of pupillometric data. First, to ensure high data yield, it is critical to familiarize the animals well to the experimental setup. A lack of patience in this step could degrade the quality of data that is eventually obtained or necessitate the repetition of several sessions to make up for the lost sessions. Second, to avoid luminance-related PD changes, it is important to perform experiments in constant illumination conditions, maintaining these conditions between sessions and subjects as much as possible. Third, to minimize the number of experimental sessions needed, it is important to perform pilot experiments to identify critical parameter ranges for dense sampling. Fourth, to minimize habituation of the animals to the stimuli, it is important to perform experiments in short sessions containing only a few presentations of deviant stimuli. An air puff may be additionally used to maintain high engagement with the auditory stimuli.

Pupil diameter (PD) can be affected by a wide number of factors and the measurement of PD that changes over time is known as pupillometry. PD is controlled by the iris sphincter muscle (involved in constriction) and the iris dilator muscle (involved in dilation). The constriction muscle is innervated by the parasympathetic system and involves cholinergic projections, whereas the iris dilator is innervated by the sympathetic system involving noradrenergic and cholinergic projections1,2,3. The best-known stimulus to induce PD changes is luminance-constriction and dilation responses of the pupil can be produced by variations in ambient light intensity2. PD also changes as a function of focal distance2. It has been known for decades, however, that PD also shows non-luminance-related fluctuations4,5,6,7. For example, changes in internal mental states can elicit transient PD changes. The pupil dilates in response to emotionally charged stimuli or increases with arousal4,5,8,9. Pupil dilation could also be related to other cognitive mechanisms, such as increased mental effort or attention10,11,12,13. Because of this relationship between pupil size variations and mental states, PD changes have been explored as a marker of clinical disorders such as schizophrenia14,15, anxiety16,17,18, Parkinson&#39;s disease19,20, and Alzheimer&#39;s disease21, among others. In animals, PD changes track internal behavioral states and are correlated with neuronal activity levels in cortical areas22,23,24,25. Pupil diameter has also been shown to be a reliable indicator of the sleep state in mice26. These PD changes related to arousal and the internal state typically occur on long time scales of the order of several tens of seconds.
In the domain of hearing research, in normal hearing as well as in hearing impaired subjects, listening effort and auditory perception have been assessed using pupillometry. These studies typically involve trained research subjects27,28,29,30 that perform various kinds of detection or recognition tasks. Because of the aforementioned relationship between arousal and PD, increased task engagement and listening effort have been shown to be correlated with increased pupil dilation responses30,31,32,33,34,35. Thus, pupillometry has been used to demonstrate that increased listening effort is expended to recognize spectrally degraded speech in normal-hearing listeners29,36. In hearing impaired listeners, such as humans with age-related hearing loss27,30,37,38,39,40,41 and cochlear implant users42,43, pupil responses also increased with decreasing speech intelligibility; however, hearing impaired listeners showed greater pupil dilation in easier listening conditions compared to normal hearing subjects27,30,37,38,39,40,41,42,43. But experiments that require the listener to perform a recognition task are not always possible - for example, in infants, or in some animal models. Thus, non-luminance related pupil responses evoked by acoustic stimuli could be a viable alternative method to assess auditory detection in these cases44,45. Earlier studies demonstrated a transient and stimulus-linked pupil dilation as part of the orienting reflex46. Later studies have demonstrated the use of stimulus-linked pupil dilations to derive frequency sensitivity curves in owls47,48. Recently, these methods have been adapted to assess sensitivity of the pupil dilation response in human infants48. Pupillometry has been shown to be a reliable and non-invasive approach to estimate auditory detection and discrimination thresholds in passively listening guinea pigs (GPs) by using a wide range of simple (tones) and complex (GP vocalizations) stimuli49. These stimulus-related PD changes typically occur at faster time scales of the order of several seconds and are linked to stimulus timing. Here, pupillometry of stimulus-related PD changes is proposed as a method to study behavioral impacts of various kinds of hearing impairment in animal models. In particular, pupillometry protocols for use in GPs, a well-established animal model of various types of auditory pathologies50,51,52,53,54,55,56 (also see reference57 for an exhaustive review) is described.
Although this technique is demonstrated in normal-hearing GPs, these methods can be easily adapted to other animal models and animal models of various auditory pathologies. Importantly, pupillometry can be combined with other non-invasive measurements such as EEG, as well as with invasive electrophysiological recordings in order to study the mechanisms underlying possible sound detection and perception deficits. Finally, this approach can also be used to establish broad similarities between human and animal models.

<table><tbody><tr><td>Analog output board</td><td>Measurement Computing Corporation, Norton, MA</td><td>PCI-DDA02/12</td><td></td></tr><tr><td>Anechoic foam</td><td>Sonex One, Pinta Acoustic, Minneapolis, MN</td><td></td><td></td></tr><tr><td>Condenser microphone</td><td>Behringer, Willich, Germany</td><td>C-2</td><td></td></tr><tr><td>Free-field microphone</td><td>Bruel &amp;amp; Kjaer, Denmark)&amp;nbsp;</td><td>Type 4940&amp;nbsp;</td><td></td></tr><tr><td>Matlab</td><td>Mathworks, Inc., Natick, MA</td><td></td><td>2018a version</td></tr><tr><td>Monocular remote camera and illuminator system</td><td>Arrington Research, Scottsdale, AZ</td><td>MCU902</td><td>Infrared LED array + camera with infrared filter</td></tr><tr><td>Multifunction I/O Device&amp;nbsp;</td><td>National Instruments, Austin, TX</td><td>PCI-6229</td><td></td></tr><tr><td>Neural interface processor</td><td>Ripple Neuro, Salt Lake City, UT</td><td>SCOUT</td><td></td></tr><tr><td>Piezoelectric motion sensor</td><td>SparkFun Electronics, Niwot, CO</td><td>SEN-10293</td><td></td></tr><tr><td>Pinch valve&amp;nbsp;</td><td>Cole-Palmer Instrument Co., Vernon Hills, IL</td><td>EW98302-02</td><td></td></tr><tr><td>Programmable attenuator</td><td>Tucker-Davis Technologies, Alachua, FL</td><td>PA5</td><td></td></tr><tr><td>Silicon Tubing</td><td>Cole-Parmer</td><td></td><td>~3 mm</td></tr><tr><td>Sound attenuating chamber</td><td>IAC Acoustics</td><td></td><td></td></tr><tr><td>Speaker full-range driver</td><td>Tang Band Speaker, Taipei, Taiwan</td><td>W4-1879</td><td></td></tr><tr><td>Stereo Amplifier</td><td>Tucker-Davis Technologies, Alachua, FL</td><td>SA1</td><td></td></tr><tr><td>Tabletop - CleanTop Optical</td><td>TMC vibration control / Ametek, Peabody, MA</td><td></td><td></td></tr><tr><td>Viewpoint software</td><td>ViewPoint, Arrington Research, Scottsdale, AZ</td><td></td><td></td></tr></tbody></table>

pupillometry to assess auditory sensation in guinea pigs

Noise exposure is a leading cause of sensorineural hearing loss. Animal models of noise-induced hearing loss have generated mechanistic insight into the underlying anatomical and physiological pathologies of hearing loss. However, relating behavioral deficits observed in humans with hearing loss to behavioral deficits in animal models remains challenging. Here, pupillometry is proposed as a method that will enable the direct comparison of animal and human behavioral data. The method is based on a modified oddball paradigm - habituating the subject to the repeated presentation of a stimulus and intermittently presenting a deviant stimulus that varies in some parametric fashion from the repeated stimulus. The fundamental premise is that if the change between the repeated and deviant stimulus is detected by the subject, it will trigger a pupil dilation response that is larger than that elicited by the repeated stimulus. This approach is demonstrated using a vocalization categorization task in guinea pigs, an animal model widely used in auditory research, including in hearing loss studies. By presenting vocalizations from one vocalization category as standard stimuli and a second category as oddball stimuli embedded in noise at various signal-to-noise ratios, it is demonstrated that the magnitude of pupil dilation in response to the oddball category varies monotonically with the signal-to-noise ratio. Growth curve analyses can then be used to characterize the time course and statistical significance of these pupil dilation responses. In this protocol, detailed procedures for acclimating guinea pigs to the setup, conducting pupillometry, and evaluating/analyzing data are described. Although this technique is demonstrated in normal-hearing guinea pigs in this protocol, the method may be used to assess the sensory effects of various forms of hearing loss within each subject. These effects may then be correlated with concurrent electrophysiological measures and post-hoc anatomical observations.

Pupillometry was performed in three male pigmented GPs, weighing ~600-1,000 g over the course of the experiments. As described in this protocol, to estimate call-in-noise categorization thresholds, an oddball paradigm was used for stimulus presentation. In the oddball paradigm, calls belonging to one category (whines) embedded in white noise at a given SNR were employed as standard stimuli (Figure 2A), and calls from another category (wheeks) embedded in white noise at the same SNR (Figure 2A) as deviant stimuli. Standard and deviant stimuli were randomly chosen, with resampling, from eight exemplars of each category. In each experimental session, stimuli were presented with high temporal regularity (Figure 2B), with at least 20 presentations of standard stimuli between deviant stimuli. Data were acquired corresponding to a particular SNR level in each experimental session. Across sessions, a broad range of clean and noisy SNRs were sampled (-24, -18, -12, -6, -3, 0, 3, 6, 12, 40 dB SNR).
The PD changes to the standard stimuli did not differ significantly from the baseline (blue line in Figure 3A). The deviant stimuli evoked robust and significantly larger PD changes than those elicited by the standard stimuli (gray lines in Figure 3A), reflecting call category discrimination. The response magnitude and the percentage of trials with statistically significant pupil responses were highest at the cleanest SNR and decreased gradually with decreasing SNR (Figure 3A,B). Using GCA, pupil responses to deviant stimuli were found to be statistically significant at SNRs above -18 dB (Figure 3C), which was taken to be the call-in-noise categorization threshold (green line in Figure 3A). The percentage of significant trials at each tested SNR level was well-fit by a psychometric function (Figure 3D). The SNR level necessary to reach the half-maximum of the psychometric curve was about -20 dB SNR (Figure 3D). Anecdotally, for this case, the reliability-based and time course-based metrics yielded similar values of call-in-noise categorization thresholds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64581/64581fig01.jpg" /> 
Figure 1: Pupillometry setup, and stimulus-evoked and motion-related PD changes. (A) The pupillometry setup with video frame images of sound evoked pupil dilation (top). The baseline PD is shown by dashed green circles. (B) An exemplar PD trace (top) and exemplar motion trace (bottom) from a single experimental session. Vertical black lines correspond to onset time deviant stimulus presentations. Red ticks correspond to automatically detected motion events. Gray horizontal dashed line corresponds to 5 SD threshold. (C) The PD changes (&#916;PD) evoked by deviant stimulus (top) and related to motion events (bottom) from one experimental session. Stimulus onset is shown by vertical black line; the detection of motion event is shown by vertical red line. Note that pupil dilation onset precedes the onset of motion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64581/64581fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64581/64581fig02v2.jpg" /> 
Figure 2: Call spectrograms and call-in-noise categorization paradigm structure. (A) Representative spectrograms of a guinea pig whine and wheek, in clean conditions and at 0- and -18-dB SNR, respectively. Noisy calls were obtained by adding white noise. (B) Structure of the oddball paradigm used to estimate call-in-noise categorization thresholds. Whine calls were randomly chosen from eight exemplars and used as standard stimuli. Wheek calls were randomly chosen from eight exemplars and used as deviants. In each experimental session, the noise was added at a different SNR level (-24, -18, -12, -6, -3, 0, 3, 6, 12 dB SNR). The calls are 1 s long and the time between stimuli is 3 s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64581/64581fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64581/64581fig03.jpg" /> 
Figure 3: Pupillometry estimates of call-in-noise detections and categorization thresholds. (A) Average pupil responses from three animals. Mean pupil responses to standard whine stimuli are represented by blue line, and shading corresponds to &#177;1 standard error of mean (s.e.m.). Gray lines and shading correspond to mean and &#177;1 s.e.m. of pupil responses evoked by deviant wheek stimuli. Gray shading intensity corresponds to SNR. Green line and shading correspond to average pupil trace at threshold SNR (about -18 dB SNR). Red vertical line corresponds to stimulus onset; orange vertical line corresponds to air puff onset; teal dashed lines correspond to GCA window (PD changes rising phase). (B) GCA fit to the rising phase of PD changes. Dots are mean pupil diameter in 100 ms time bins, whiskers correspond to &#177;1 s.e.m. Solid lines correspond to mixed-effects model fits. Line colors as in A. (C) GCA weight estimates. Weights of the intercept is in blue, slope is in red, and acceleration is in purple. Whiskers correspond to &#177;1 s.e.m. Asterisks show statistically significant regression weights (linear hypothesis test on linear regression model coefficients). (D) Psychometric function fit to the percent of trials with significant PD changes elicited by the deviant stimulus as a function of SNR. Whiskers correspond to &#177;1 s.e.m. Note that 50% of the maximum is reached at about -20 dB SNR (green dashed line). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64581/64581fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Pupillometry to Assess Auditory Sensation in Guinea Pigs at JoVE.com

Pupillometry to Assess Auditory Sensation in Guinea Pigs

Pupillometry, a simple and non-invasive technique, is proposed as a method to determine hearing-in-noise thresholds in normal hearing animals and animal models of various auditory pathologies.

Noise exposure is a leading cause of sensorineural hearing loss. Animal models of noise-induced hearing loss have generated mechanistic insight into the ...

pupillometry-to-assess-auditory-sensation-in-guinea-pigs

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.7K Views.  University of Pittsburgh. Pupillometrie kan worden gebruikt om complexe geluidsherkenningsdrempels te schatten en kan in combinatie met andere methoden worden gebruikt om de effecten van verschillende vormen van gehoorverlies op die drempels te bepalen. Deze techniek kan worden gebruikt om gegevens van dezelfde modaliteit van mensen en van ongetrainde dieren te karakteriseren. Het is relatief eenvoudig in te stellen en minimaal invasief.Gedrags- en perceptiemetingen zijn moeilijk te verkrijgen in diermodellen v...

Video: Pupillometry to Assess Auditory Sensation in Guinea Pigs

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

A Low Cost Setup for Behavioral Audiometry in Rodents

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 na&iuml;ve 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 na&iuml;ve, 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).

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 ...

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

Biology

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

Medicine

Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for inducing CHL in rodents. The protocol demonstrates CHL by tympanic membrane puncture. Verification of CHL surgery was by otoscope examination and behavioral assessment by clap startle response, both replicable and reliable, and are simple methods to demonstrate hearing loss has occurred. The simple CHL procedure is advantageous due to its reproducibility and flexibility to different pursuits in hearing loss research. The limitations of inducing CHL by a surgical approach are associated with the learning curve to perform the surgical procedure and confidence in audiological examination. Inducing a hearing impairment by CHL allows one to readily study the neural manifestations and behavioral outcomes of hearing loss.

Here, we present a protocol to establish a replicable conductive hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle.

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for ...

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

The Miniature Pig: A Large Animal Model for Cochlear Implant Research

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, no standard model exists for investigating the electrophysiology and histopathology in patients or animal models with a CI or for evaluating new models of electrode arrays. A large animal model with cochlea characteristics similar to those of humans may provide a research and evaluation platform for advanced and modified arrays before their use in humans. 
To this end, we established standard CI methods with Bama mini-pigs, whose inner ear anatomy is highly similar to that of humans. Arrays designed for human use were implanted into the mini pig cochlea through a round window membrane, and a surgical approach followed that was similar to that used for human CI recipients. Array insertion was followed by evoked compound action potential (ECAP) measurements to evaluate the function of the auditory nerve. This study describes the preparation of the animal, surgical steps, array insertion, and intraoperative electrophysiological measurements. 
The results indicated that the same CI used for humans could be easily implanted in mini-pigs via a standardized surgical approach and yielded similar electrophysiological outcomes as measured in human CI recipients. Mini-pigs could be a valuable animal model to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches before applying them to human beings.

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

Cochlear implants (CI) are the most effective method to treat people with severe-to-profound sensorineural hearing loss. Although CIs are used worldwide, ...

Recording Auditory Brainstem Responses in a Rat Pup

To begin this procedure, place the anesthetized animal in a polyethylene foam mold to immobilize the animal&#39;s body. Then, place the animal on an anti-vibration table in a sound-attenuating room. Keep the animal&#39;s body temperature at 37.5 degrees Celsius with a heating pad.
Next, wipe the head area with 70% ethanol. Make a 1 to 2-millimeter incision ventrolaterally to the external pinna, to place the reference electrode or ground electrode. Then, place a subdermal recording electrode over the skull vertex.
Using the function generator, generate calibrated tone bursts of various frequencies and intensities. Deliver the sound stimuli through an electrostatic speaker located 10 centimeters away from the head of the animal.
To obtain the auditory brainstem responses, the sound elicited potentials are filtered, amplified, and averaged using a multifunction processor. The 40-minute ABR recording is monitored online and stored for offline analysis.

Pupillometry to Assess Auditory Sensation in Guinea Pigs

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