Auditory Evoked Potential Thresholds Research Articles

Age-related loss of auditory sensitivity in the zebrafish (Danio rerio)

Age-related hearing loss (ARHL), also known as presbycusis, is a widespread and debilitating condition impacting many older adults. Conventionally, researchers utilize mammalian model systems or human cadaveric tissue to study ARHL pathology. Recently, the zebrafish has become an effective and tractable model system for a wide variety of genetic and environmental auditory insults, but little is known about the incidence or extent of ARHL in zebrafish and other non-mammalian models. Here, we evaluated whether zebrafish exhibit age-related loss in auditory sensitivity. The auditory sensitivity of adult wild-type zebrafish (AB/WIK strain) from three adult age subgroups (13-month, 20-month, and 37-month) was characterized using the auditory evoked potential (AEP) recording technique. AEPs were elicited using pure tone stimuli (115–4500 Hz) presented via an underwater loudspeaker and recorded using shielded subdermal metal electrodes. Based on measures of sound pressure and particle acceleration, the mean AEP thresholds of 37-month-old fish [mean sound pressure level (SPL) = 122.2 dB ± 2.2 dB SE re: 1 μPa; mean particle acceleration level (PAL) = -27.5 ± 2.3 dB SE re: 1 ms−2] were approximately 9 dB higher than that of 20-month-old fish [(mean SPL = 113.1 ± 2.7 dB SE re: 1 μPa; mean PAL = -37.2 ± 2.8 dB re: 1 ms−2; p = 0.007)] and 6 dB higher than that of 13-month-old fish [(mean SPL = 116.3 ± 2.5 dB SE re: 1 μPa; mean PAL = -34.1 ± 2.6 dB SE re: 1 ms−2; p = 0.052)]. Lowest AEP thresholds for all three age groups were generally between 800 Hz and 1850 Hz, with no evidence for frequency-specific age-related loss. Our results suggest that zebrafish undergo age-related loss in auditory sensitivity, but the form and magnitude of loss is markedly different than in mammals, including humans. Future work is needed to further describe the incidence and extent of ARHL across vertebrate groups and to determine which, if any, ARHL mechanisms may be conserved across vertebrates to support meaningful comparative/translational studies.

Electrophysiological Threshold Evaluation in Infants with and Without Risk Indicators for Hearing Loss

Introduction: The brainstem auditory evoked potential is used to determine the electrophysiological threshold and assessing the integrity of the auditory system. This test is sensitive to the auditory nerve maturation and brainstem; therefore, the electrophysiological threshold may change throughout child development. Objective: to evaluate the changes in the electrophysilogical threshold of brainstem auditory evoked potential in two months of follow-up. Methods: A single non-concurrent cohort study was performed in a public hospital during January 2013 to January 2015. The brainstem auditory evoked potential was performed in all neonates in two moments with electrophysiological thresholds measured in the both ears, whose results were categorized into degree: mild, moderate, severe and profound. Results: Forty-three infants of both genders, 11 with prematurity and 14 with risk indicators for hearing loss participated in the study. In the first brainstem auditory evoked potential, with an average age of two months, 34 infants had abnormal results in the right ear and 31 in the left ear. In the second brainstem auditory evoked potential, with na average age of four months, normalization was observed in 38% of the results obtained in the right ear and in 42% on the left ear. This significant change in brainstem auditory evoked potential thresholds was found in those with mild and moderate degrees. Conclusion: There were changes in the electrophysiological brainstem auditory evoked potential thresholds in infants with a mild and moderate degree, while those with a severe and profound degree the results were permanet at two months of follow-up.

Development of an S3/SC1 standard for auditory evoked potential hearing tests in toothed whales

The National Marine Fisheries service regulates the impact of anthropogenic ocean noise to marine mammals. Other government and commercial groups that produce ocean noise are subject to regulation. Both groups desire greater knowledge of hearing in marine mammals to better predict, mitigate, and regulate noise exposure. Auditory evoked potential (AEP) hearing tests have become a primary method by which hearing tests are performed in toothed whales (e.g., dolphins, porpoises). The synthesis of AEP data to address noise impacts to marine mammals is limited, in part, because of the variability between AEP thresholds measured by different researchers and laboratories using different methodologies. Methodological differences exist for AEP detection, threshold estimation, calibration, and stimulus delivery. Threshold variability could be reduced through standardization of AEP hearing test methods, which is desired by regulatory agencies and sound producers. An S3/SC1 standard for AEP hearing test methods in toothed whales has been initiated to address this need. The draft standard will be submitted to ANSI for consideration by the end of 2017. Adoption of the standard internationally would further improve threshold comparability by ensuring researchers globally utilize the same methods. The process should increase AEP data reliability for purposes of addressing ocean noise issues.

Auditory evoked potential audiograms in post-settlement stage individuals of coral reef fishes

Just after the reef colonization, fish species could use the acoustic cue to settle on different suitable habitats. In the present study, we used the auditory evoked potential (AEP) technique to measure and compare the detection abilities in five coral reef fish species, with some of these species that are found in the same habitat. We also examined the effect of fish size on sensitivity at the species level. All studied species except one showed size-related changes in sensitivity characterized by either a decrease (i.e. higher AEP thresholds) or an increase (i.e. lower AEP thresholds) in detection abilities with increasing size. The interspecific comparison of audiograms revealed that some species are more sensitive than others in terms of sound pressure level and frequency detection.Overall, this study indicates that the AEP threshold and the frequency bandwidth at early life stages may vary between and within fish species. The detection abilities are different in fish species that are not phylogenetically related, which might suggest that the establishment of their capabilities is not necessarily related to the reef conquest.

Ontogenetic development of the auditory sensory organ in zebrafish (Danio rerio): changes in hearing sensitivity and related morphology.

Zebrafish (Danio rerio) is an important model organism in hearing research. However, data on the hearing sensitivity of zebrafish vary across different reports. In the present study, the hearing sensitivity of zebrafish was examined by analysing the auditory evoked potentials (AEPs) over a range of total lengths (TLs) from 12 to 46 mm. Morphological changes in the hair cells (HCs) of the saccule (the main auditory end organ) and their synapses with primary auditory neurons were investigated. The AEPs were detected up to a much higher frequency limit (12 kHz) than previously reported. No significant difference in the frequency response range was observed across the TL range examined. However, the AEP thresholds demonstrated both developmental improvement and age-related loss of hearing sensitivity. The changes in hearing sensitivity were roughly consistent with the morphological changes in the saccule including (1) the number and density of HCs, (2) the organization of stereocilia, and (3) the quantity of a main ribbon protein, Ribeye b. The results of this study established a clear baseline for the hearing ability of zebrafish and revealed that the changes in the saccule contribute to the observed changes in TL (age)-related hearing sensitivity.

Hearing threshold measurements of five stranded short-finned pilot whales (Globicephala macrorhynchus)

On May 5, 2011, 23 short-finned pilot whales, Globicephala macrorhynchus, were stranded along the coastline near Cudjoe Key, FL. Five animals (two adult females, two juvenile females, and an adult male) were transported to a rehabilitation facility in Key Largo, FL. Auditory evoked potentials (AEPs) were recorded in response to amplitude modulated tone pips modulated at 1000 Hz. AEP thresholds were determined at 10, 20, 40, 80, and 120 kHz for the four females. However, the adult male was euthanized prior to testing. Short-finned pilot whales had peak sensitivity at lower frequencies than other odontocetes such as bottlenose dolphins. Greatest sensitivity was around 40 kHz for all whales, while thresholds for the two adult females were 25-61 dB higher at 80 kHz than the juveniles. Click evoked potentials were similar between the four whales and comparable to other echolocating odontocetes. Click evoked potential data from a fifth short-finned pilot whale that had stranded in Curacao showed no response. These findings add to the limited database of pilot whale (short- and long-finned) hearing studies, of which there are only two others [Schlundt et al. (2011). J. Acoust. Soc. Am. 129, 1111-1116 and Pacini et al. (2010). J. Exp. Biol. 213, 3138-3143].

Underwater psychophysical audiogram of a young male California sea lion (Zalophus californianus)

Auditory evoked potential (AEP) data are commonly obtained in air while sea lions are under gas anesthesia; a procedure that precludes the measurement of underwater hearing sensitivity. This is a substantial limitation considering the importance of underwater hearing data in designing criteria aimed at mitigating the effects of anthropogenic noise exposure. To determine if some aspects of underwater hearing sensitivity can be predicted using rapid aerial AEP methods, this study measured underwater psychophysical thresholds for a young male California sea lion (Zalophus californianus) for which previously published aerial AEP thresholds exist. Underwater thresholds were measured in an aboveground pool at frequencies between 1 and 38 kHz. The underwater audiogram was very similar to those previously published for California sea lions, suggesting that the current and previously obtained psychophysical data are representative for this species. The psychophysical and previously measured AEP audiograms were most similar in terms of high-frequency hearing limit (HFHL), although the underwater HFHL was sharper and occurred at a higher frequency. Aerial AEP methods are useful for predicting reductions in the HFHL that are potentially independent of the testing medium, such as those due to age-related sensorineural hearing loss.

Underwater hearing in the loggerhead turtle (Caretta caretta): a comparison of behavioral and auditory evoked potential audiograms

The purpose of this study was to compare underwater behavioral and auditory evoked potential (AEP) audiograms in a single captive adult loggerhead sea turtle (Caretta caretta). The behavioral audiogram was collected using a go/no-go response procedure and a modified staircase method of threshold determination. AEP thresholds were measured using subdermal electrodes placed beneath the frontoparietal scale, dorsal to the midbrain. Both methods showed the loggerhead sea turtle to have low frequency hearing with best sensitivity between 100 and 400 Hz. AEP testing yielded thresholds from 100 to 1131 Hz with best sensitivity at 200 and 400 Hz (110 dB re. 1 μPa). Behavioral testing using 2 s tonal stimuli yielded underwater thresholds from 50 to 800 Hz with best sensitivity at 100 Hz (98 dB re. 1 μPa). Behavioral thresholds averaged 8 dB lower than AEP thresholds from 100 to 400 Hz and 5 dB higher at 800 Hz. The results suggest that AEP testing can be a good alternative to measuring a behavioral audiogram with wild or untrained marine turtles and when time is a crucial factor.

Hearing threshold measurements using auditory evoked potentials of four stranded short-finned pilot whales (Globicephala macrorhynchus) in Key Largo, FL

Approximately 26 short-finned pilot whales, Globicephala macrorhynchus, stranded in Cudjoe Key, FL, on May 5, 2011. Four animals, two adult and two juvenile females, were transported to a rehabilitation facility in Tarpon Basin, FL. Auditory evoked potentials (AEPs) were recorded in response to amplitude modulated tone pips modulated at 1000 Hz. AEP thresholds were determined at 10, 20, 40, 80, and 120 kHz for all the four animals. Audiograms were similar to previous findings in pilot whale hearing tests. Short-finned pilot whales have a lower peak sensitivity than other odontocetes such as bottlenose dolphins. Greatest sensitivity was 40 kHz for all whales, while thresholds for the two adults were 25–61 dB higher at 80 kHz than those for the juveniles tested. Click evoked potentials were similar between the four whales and comparable to other echolocating odontocetes. Five total pilot whales have been tested during two separate stranding events; the previously tested juvenile male was found to have profound hearing loss (Mann et al., 2010; Schlundt et al., 2011). These findings add to the limited database of pilot whale (short- and long-finned) hearing studies, of which there are only two others (Schlundt et al., 2011; Pacini et al., 2010).

Hearing ability of the lined seahorse (Hippocampus erectus).

The lined seahorse (Hippocampus erectus), a member of the family Syngnathidae (pipefishes and seahorses), occurs in coastal waters of the western Atlantic, Gulf of Mexico, and Caribbean Sea. This harmless species is listed as “vulnerable” on the International Union for the Conservation of Nature (IUCN) List of Threatened Species, meaning that it has a high risk of extinction in the medium‐term future primarily due to declines in population from targeted catch, bycatch, and degradation of habitat. However, because little is known about hearing in Syngnathids, it is difficult to assess potential effects of anthropogenic sound on these animals. In this study auditory evoked potentials (AEPs) were measured in four H. erectus specimens to determine hearing thresholds and bandwidth. Thresholds were determined using the staircase method with exposures to tone bursts between 50 Hz and 21.6 kHz. At low frequencies H. erectus had AEP thresholds similar to other bony fishes; however, at frequencies above 2 kHz its auditory sensitivity was similar to that of clupeiform species which detect ultrasound [Mann et al., J. Acoust. Soc. Am. (2001)]. The results of this study provide hearing data that can be used to understand potential effects of human‐generated noise on Hippocampus erectus and other seahorse species.

Does speaker presentation affect auditory evoked potential thresholds in goldfish?

The auditory evoked potential (AEP) recording technique has proved to be a very versatile and successful approach in studying auditory sensitivities in fishes. The AEP protocol introduced by Kenyon, Ladich and Yan in 1998 using an air speaker with the fish positioned at the water surface gave auditory thresholds in goldfish very close to behavioural values published before. This approach was subsequently modified by several laboratories, raising the question whether speaker choice (air vs. underwater) or the position of subjects affect auditory threshold determination. To answer these questions, the hearing specialist Carassius auratus was measured using an air speaker, an underwater speaker and alternately positioning the fish directly at or 5 cm below the water surface. Mean hearing thresholds obtained using these 4 different setups varied by 5.6 dB, 3.7 dB and 4 dB at 200 Hz, 500 Hz and 1000 Hz, respectively. Accordingly, pronounced differences in AEP thresholds in goldfish measured in different laboratories reflect other factors than speaker used and depth of the test subjects, namely variations in threshold definition, background noise, population differences, or calibration errors.

Barging Effects on Sensory Systems of Chinook Salmon Smolts

AbstractTo avoid mortality caused by passage through dam turbines and spillways, juvenile Chinook salmon Oncorhynchus tshawytscha are annually transported downstream by barge through the federal hydropower system on the Snake and Columbia rivers. Survival of transported fish is higher than that of in‐river migrants; however, transported fish experience higher rates of postrelease mortality. Increased mortality could result from a decrease in the ability to detect or avoid predators due to stressors associated with the barge environment. This study examined the effects of barging on juvenile Chinook salmon olfaction and auditory function, two sensory systems involved in predator detection. We focused on dissolved metals known to be toxic to the salmon olfactory system and on the level of noise from the barge, which could impair the auditory system. Experimental groups included animals collected (1) before barge loading (control group), (2) at the Bonneville Dam bypass system (migrant fish), (3) immediately after barge transport, and (4) within 7 d postbarging and at or after 7 d postbarging. Measured concentrations of dissolved metals from the water within the barge were below established water quality criteria for the protection of aquatic life. Moreover, ultrastructural examination of the olfactory epithelium surface showed no evidence of injury to olfactory sensory neurons. Noise in the barge holding tanks had levels up to 136 dB referenced to 1 μPa (root mean square) with primary energy below 400 Hz. Auditory sensitivity was measured using the auditory‐evoked potentials (AEP) technique. We found a small but statistically significant threshold shift for fish collected within 7 d postbarging, while in the 7‐d‐and‐later postbarging group the AEP thresholds were similar to the control. Our findings indicate that the olfactory systems of transported Chinook salmon are intact and probably functional, while the auditory sensitivities are compromised with probable recovery.

Progress in clinical hearing evaluation of bottlenose dolphins (Tursiops truncatus).

It has long been recognized that the need to train experimental subjects is a major obstacle to large scale behavioral hearing studies of marine mammals. The time and level of access required for behavioral audiometry have not only prevented hearing assessment in wild individuals but have also limited attempts to assess auditory system fitness in captive populations. Ridgway and co‐workers of electrophysiological methods were quick to realize that auditory evoked potential (AEP) measurements could be used to assess marine mammal hearing without lengthy training. Ridgway performed some of the earliest work to characterize dolphin evoked potentials and to develop fast techniques for threshold measurement. In addition to hearing assessment in larger whales not routinely kept in captivity, Ridgway has also strived to develop techniques that would allow routine clinical hearing assessment of captive animals. This talk reviews the development of AEP techniques for rapid large‐scale hearing assessment in dolphins at the US Navy Marine Mammal Program. Topics range from Ridgway’s early work on dolphin AEP threshold measurements to recent efforts to acquire longitudinal audiometric data from a large population of dolphins. [Work supported by ONR.]

Arbitrary evoked potentials: using AEPs to measure hearing in fishes

Auditory evoked potential (AEP) techniques have become commonly used to measure hearing of a wide variety of animals. However, there is no standard way of defining a threshold from auditory evoked potential data. There are two arbitrary factors affecting the precision of AEP data: the number of sweeps averaged together to detect the AEP, and the method of calculating a threshold from the AEP data. While these arbitrary factors do not invalidate comparative studies where these variables are standardized, different laboratories use different techniques. We describe sources of variation in estimating thresholds using evoked potential techniques using the goldfish as an example. There was little variation in AEP thresholds due to variation in electrode placement or in the method of arbitrarily assigning an AEP threshold. The largest variation was due to differences in the number of sweeps averaged. Under controlled conditions using the same goldfish in the same test tank with the same number of sweeps averaged, AEP's better predicted behavioral thresholds at high frequencies than low frequencies. Since averaging reduces uncorrelated background noise (neural and electrical), it should theoretically be possible to obtain AEP measurements below behavioral thresholds with enough sweeps averaged together.

Sound production and spectral hearing sensitivity in the Hawaiian sergeant damselfish, Abudefduf abdominalis

Sounds provide important signals for inter- and intraspecific communication in fishes, but few studies examine fish acoustic behavior in the context of coevolution of sound production and hearing ability within a species. This study characterizes the acoustic behavior in a reproductive population of the Hawaiian sergeant fish, Abudefduf abdominalis, and compares acoustic features to hearing ability, measured by the auditory evoked potential (AEP) technique. Sergeant fish produce sounds at close distances to the intended receiver (<or=1-2 body lengths), with different pulse characteristics that are associated primarily with aggression, nest preparation and courtship-female-visit behaviors. Energy peaks of all sounds were between 90 and 380 Hz, whereas courtship-visit sounds had a pulse repetition rate of 125 Hz with harmonic intervals up to 1 kHz. AEP threshold, which is probably higher than the behavioral threshold, indicates best sensitivity at low frequencies (95-240 Hz), with the lowest threshold at 125 Hz (123-127 dB(rms) re: 1 microPa). Thus, sound production and hearing in A. abdominalis are closely matched in the frequency domain and are useful for courtship and mating at close distances. Measured hearing thresholds did not differ among males and females during spawning or non-spawning periods, which indicates a lack of sex differences and seasonal variation in hearing capabilities. These data provide the first evidence that Abudefduf uses true acoustic communication on a level similar to that of both more derived (e.g. Dascyllus, Chromis) and more basal (e.g. Stegastes) soniferous pomacentrids. This correlation between sound production and hearing ability is consistent with the sensory drive model of signal evolution in which the sender and receiver systems coevolve within the constraints of the environment to maximize information transfer of acoustic signals.

Simultaneously measured behavioral and electrophysiological hearing thresholds in a bottlenose dolphin (Tursiops truncatus)

Dolphin auditory thresholds obtained via evoked potential audiometry may deviate from behavioral estimates by 20 dB or more. Differences in the sound source, stimulus presentation method, wave form, and duration may partially explain these discrepancies. To determine the agreement between behavioral and auditory evoked potential (AEP) threshold estimates when these parameters are held constant, behavioral and AEP hearing tests were simultaneously conducted in a bottlenose dolphin. Measurements were made in-air, using sinusoidal amplitude-modulated tones continuously projected via a transducer coupled to the pan region of the dolphin's lower jaw. Tone trials were presented using the method of constant stimuli. Behavioral thresholds were estimated using a 50% correct detection. AEP thresholds were based on the envelope following response and 50% correct detection. Differences between AEP and behavioral thresholds were within +/-5 dB, except at 10 kHz (12 dB), 20 kHz (8 dB), 30 kHz (7 dB), and 150 kHz (24 dB). In general, behavioral thresholds were slightly lower, though this trend was not significant. The results demonstrate that when the test environment, sound source, stimulus wave form, duration, presentation method, and analysis are consistent, the magnitude of the differences between AEP and behavioral thresholds is substantially reduced.

A comparison of underwater hearing sensitivity in bottlenose dolphins (Tursiops truncatus) determined by electrophysiological and behavioral methods

Variable stimulus presentation methods are used in auditory evoked potential (AEP) estimates of cetacean hearing sensitivity, each of which might affect stimulus reception and hearing threshold estimates. This study quantifies differences in underwater hearing thresholds obtained by AEP and behavioral means. For AEP estimates, a transducer embedded in a suction cup (jawphone) was coupled to the dolphin's lower jaw for stimulus presentation. Underwater AEP thresholds were obtained for three dolphins in San Diego Bay and for one dolphin in a quiet pool. Thresholds were estimated from the envelope following response at carrier frequencies ranging from 10 to 150 kHz. One animal, with an atypical audiogram, demonstrated significantly greater hearing loss in the right ear than in the left. Across test conditions, the range and average difference between AEP and behavioral threshold estimates were consistent with published comparisons between underwater behavioral and in-air AEP thresholds. AEP thresholds for one animal obtained in-air and in a quiet pool demonstrated a range of differences of -10 to 9 dB (mean = 3 dB). Results suggest that for the frequencies tested, the presentation of sound stimuli through a jawphone, underwater and in-air, results in acceptable differences to AEP threshold estimates.

Comparison of in-air evoked potential and underwater behavioral hearing thresholds in four bottlenose dolphins ( Tursiops truncatus )

Traditional behavioral techniques for hearing assessment in marine mammals are limited by the time and access required to train subjects. Electrophysiological methods, where passive electrodes are used to measure auditory evoked potentials (AEPs), are attractive alternatives to behavioral techniques; however, there have been few attempts to compare AEP and behavioral results for the same subject. In this study, behavioral and AEP hearing thresholds were compared in four bottlenose dolphins. AEP thresholds were measured in-air using a piezoelectric sound projector embedded in a suction cup to deliver amplitude modulated tones to the dolphin through the lower jaw. Evoked potentials were recorded noninvasively using surface electrodes. Adaptive procedures allowed AEP hearing thresholds to be estimated from 10 to 150 kHz in a single ear in about 45 min. Behavioral thresholds were measured in a quiet pool and in San Diego Bay. AEP and behavioral threshold estimates agreed closely as to the upper cutoff frequency beyond which thresholds increased sharply. AEP thresholds were strongly correlated with pool behavioral thresholds across the range of hearing; differences between AEP and pool behavioral thresholds increased with threshold magnitude and ranged from 0 to + 18 dB.

Behavioral and auditory evoked potential audiograms of a false killer whale (Pseudorca crassidens)

Behavioral and auditory evoked potential (AEP) audiograms of a false killer whale were measured using the same subject and experimental conditions. The objective was to compare and assess the correspondence of auditory thresholds collected by behavioral and electrophysiological techniques. Behavioral audiograms used 3-s pure-tone stimuli from 4 to 45 kHz, and were conducted with a go/no-go modified staircase procedure. AEP audiograms used 20-ms sinusoidally amplitude-modulated tone bursts from 4 to 45 kHz, and the electrophysiological responses were received through gold disc electrodes in rubber suction cups. The behavioral data were reliable and repeatable, with the region of best sensitivity between 16 and 24 kHz and peak sensitivity at 20 kHz. The AEP audiograms produced thresholds that were also consistent over time, with range of best sensitivity from 16 to 22.5 kHz and peak sensitivity at 22.5 kHz. Behavioral thresholds were always lower than AEP thresholds. However, AEP audiograms were completed in a shorter amount of time with minimum participation from the animal. These data indicated that behavioral and AEP techniques can be used successfully and interchangeably to measure cetacean hearing sensitivity.

Ground-truthing evoked potential measurements against behavioral conditioning in the goldfish, Carassius auratus

Auditory evoked potentials (AEPs) have become commonly used to measure hearing thresholds in fish. However, it is uncertain how well AEP thresholds match behavioral hearing thresholds and what effect variability in electrode placement has on AEPs. In the first experiment, the effect of electrode placement on AEPs was determined by simultaneously recording AEPs from four locations on each of 12 goldfish, Carassius auratus. In the second experiment, the hearing sensitivity of 12 goldfish was measured using both classical conditioning and AEP’s in the same setup. For behavioral conditioning, the fish were trained to reduce their respiration rate in response to a 5 s sound presentation paired with a brief shock. A modified staircase method was used in which 20 reversals were completed for each frequency, and threshold levels were determined by averaging the last 12 reversals. Once the behavioral audiogram was completed, the AEP measurements were made without moving the fish. The recording electrode was located subdermally over the medulla, and was inserted prior to classical conditioning to minimize handling of animal. The same sound stimuli (pulsed tones) were presented and the resultant evoked potentials were recorded for 1000–6000 averages. AEP input–output functions were then compared to the behavioral audiogram to compare techniques for estimating behavioral thresholds from AEP data.