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.
