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.

For all experimental procedures, obtain approval from the Institutional Animal Care and Use Committee (IACUC) and adhere to NIH Guidelines for the care and use of laboratory animals. In the United States of America, GPs are additionally subject to United States Department of Agriculture (USDA) regulations. All the procedures in this protocol were approved by the University of Pittsburgh IACUC and adhered to NIH Guidelines for the care and use of laboratory animals. For this experiment, three male wildtype, pigmented GPs ...

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 an...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; Kjaer, Denmark)</td>
			<td>Type 4940&#160;</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</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</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 (<strong ...

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 can be used to estimate complex sound recognition thresholds, and combined with other methods can be used to determine the effects of various forms of hearing loss on those thresholds. This technique can be used to characterize data of the same modality from humans and from untrained animals. It is relatively easy to set up and minimally invasive.Behavior and perception measures are difficult to obtain in animal models of hearing loss. This method is one way to quantify complex sound recognition behaviors in animals. As we do all animal behavioral experiments patience is the key.Take the time to acclimate the animal to the experimental setup. Keep experimental sessions short and monitor the animal closely. To begin mount a calibrated loud speaker onto the sound attenuated chamber wall at an equal height to the position where the animal will be placed.For free field stimulus delivery, place the animal in the enclosure ensuring that large body movements are not possible and fix the head of the animal to the rigid frame. Place a piezoelectric sensor beneath the enclosure in order to detect and record animal movements. Then position the pupil imaging camera at a distance of 25 centimeters from the subject's eye.To set up the air puff, use a holder attached to the tabletop to place a pipette tip at around 15 centimeters in front of the animal's snout. Connect a silicone tube of around three millimeter diameter to the pipette tip and connect the tube to a regulated air cylinder. Keep the cylinder air pressure between 20 and 25 psi.And pass the tube through a pinch valve to control the timing and duration of the air puff, using a computer controlled relay. Illuminate the eye with an infrared LED array placed at around 10 centimeters distance. Use white LED lighting at an intensity of around 2000 candelas per square meter to illuminate the imaged eye and bring the baseline pupil diameter to around 3.5 millimeters.Open the pupil acquisition software and acquire the video of the pupil using a camera with a 16 millimeter lens with a spacial resolution of a 0.15 degrees visual angle and an infrared filter placed at a 25 centimeter distance from the imaged eye. Ensure that the eye is centered in the imaged area. Regulate the aperture and focus of the camera as well as the IR level until the outline of the imaged pupil is in sharp focus.In the pupil acquisition software, define the area of interest containing the pupil by selecting a rectangular area with the mouse. Then use the controls panel to adjust the brightness and contrast of the acquired video. Set the scan density to five and adjust the threshold such that the ellipse fit closely matches the outline of the pupil in the video.Using the neural interface processor software, acquire and save the analog signal from the pupil diameter or PD trace, the voltage trace from the piezoelectric sensor recording motion, the stimulus delivery times, and the air puff delivery times. Select eight different exemplars of Guinea pig vocalizations of similar lengths from two different categories of vocalizations. For example, wheek calls and whine calls.One category will serve as standard stimuli and the other category will serve as the oddball or deviant stimuli. To generate one second long standard and deviant stimuli embedded in noise at different signal-to-noise-ratio or SNR levels, add white noise of equal length to the calls. The range of SNRs sampled in this experiment is between minus 24 and plus 40 decibels.For each session, prepare a pseudorandom stimulus presentation sequence that contains standard stimuli greater than 90%of the time. Ensure that between deviant stimuli there are at least 20 to 40 trials with standard stimuli. Use a fixed stimulus intensity for all stimulus presentations.Present the stimuli with high temporal regularity. To maintain the animal's engagement with the stimuli and to minimize habituation, optionally deliver a brief air puff after the deviant stimulus. Ensure that the onset of the air puff is sufficiently separated from the stimulus duration so that stimulus evoked pupil dilation responses.Reach a peak before air puff induced blink artifacts. Run the code pupil_avg_JOVE_m and select the data file from a single session in the pop-up dialogue to perform motion detection and trial exclusion for every session. Then run the same code and select all the data files to be analyzed in the pop-up dialogue.To remove eye blink artifacts, pre-process the data, and obtain the average pupil dilation to each stimulus across sessions. Average the stimulus evoked pupil diameter changes for each stimulus condition across sessions within each animal and then across animals to generate the mean pupil dilation response to each stimulus condition. Vertically concatenate all the outputs from the code pupil_avg_JOVE.m or all the sessions animals, SNRs, and attenuations to construct a matrix containing the columns, animal ID, SNR, sound level, and pupil diameter values. For each SNR, plot the weights corresponding to the intercept to visualize the results. Do the same for linear and quadratic terms.Put all the session wise percentage of trials with significant pupil changes into each cell of a cell array where the cells are arranged from lower to higher SNR. Using the code pupil_threshold_estimate_JOVE_m, estimate the call in noise categorization threshold. Average pupil responses from three animals are shown in this figure.Mean pupil responses to standard whine stimuli are represented by the blue line and shading corresponds to plus minus one standard error of mean. Gray lines and shading correspond to mean and plus minus one standard error of mean of pupil responses evoked by deviant wheek stimuli. Gray shading intensity corresponds to SNR.Green line and shading correspond to the average pupil trace at threshold SNR. The red vertical line corresponds to stimulus onset. The orange vertical line corresponds to air puff onset and the teal dashed lines correspond to GCA window.The psychometric function fit to the percent of trials with significant pupil diameter changes elicited by the deviant stimulus as a function of SNR is shown in this figure. Whiskers correspond to plus minus one standard error of mean. Note that 50%of the maximum is reached at about minus 20 decibels SNR.The acquired high quality data, it is important to acclimate the animal well to the experimental setup and maintain constant elimination conditions. Keep the experiment session short to minimize animal's habitation to the deviant stimuli. EEG, or electrophysiological recordings combined with pupillometry would generate additional insight into the neural deficits, underlying call-in-noise categorization deficits in hearing impaired animals.

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1.6K Views.  University of Pittsburgh. Pupillometry can be used to estimate complex sound recognition thresholds, and combined with other methods can be used to determine the effects of various forms of hearing loss on those thresholds. This technique can be used to characterize data of the same modality from humans and from untrained animals. It is relatively easy to set up and minimally invasive.Behavior and perception measures are difficult to obtain in animal models of hearing loss. This method is one way to quantify co...