Patent Application
20180085034
Quick and accurate assessment of auditory thresholds is a prerequisite for experimental manipulations in the field of auditory research. To date, a variety of protocols have been used to determine audiometric thresholds. Auditory brainstem responses, behavioral audiograms, and startle reflex audiometry have each been used to assess hearing, yet each has limitations which should be taken into account when interpreting data related to threshold shifts and overall cochlear damage following sound or chemical lesions.
Perhaps the most ubiquitous test used to assess hearing performance is the auditory brainstem response. Rapid assessment of auditory brainstem circuitry makes ABR a good candidate for detecting gross changes in the auditory system. It is known that following an auditory insult, ABRs reliably identify elevated thresholds. This temporary threshold shift is thought to result from swelling of cochlear nerve terminals which is present for days after exposure. When measured with ABRs, thresholds return to baseline soon after noise exposure. Robertson D, Hearing Research 9, 263-278 (1983). However, recent work has clearly demonstrated that this measure does not account for the trauma-induced damage to ribbon synapses. Kujawa S G, Liberman M C, J Neurosci 29, 14077-14085 (2009). ABR wave one amplitudes have been shown to more accurately represent suprathreshold hearing loss, as they correlate strongly with ribbon synapse denervation following sound exposure. Liberman et al., J Neurosci 34(13): 4599-4607 (2014). However, the ABR methodology is not without some caveats. Immediately following sound exposure, ABR thresholds are often elevated so strongly that they cannot be accurately measured which also precludes wave one amplitudes from being assessed. Furthermore, ABR's are typically collected under anesthesia and are challenging to measure in animals with larger body mass. Lastly, some have contended that while ABRs thresholds are useful in detecting noise-induced damage to the auditory brainstem, they do not provide any perceptual indications of hearing loss. Alternatively, others have stated that ABRs can closely approximate behavioral thresholds in humans, yet ABRs are known to be less precise. For these reasons, it is useful to explore alternatives for hearing assessment.
Many years ago it was found that prepulse modulation of the acoustic startle reflex (ASR) could be employed to assess behavioral response thresholds. Fechter et al., J. Acoustic. Soc. Am. 84(1), 179-185 (1988). Prepulse inhibition, a decrease of ASR magnitude when a preceding weaker sound (prepulse) is presented before the startle, has been used for over half a century to objectively measure complex neurological systems. Hoffman H S, Searle J L, J Comp Physiol Psychol., 60(1), 53-581965 (1965); Gerrard R L, Ison J R, J Experimental Psych: Animal Behavior Processes 16(1): 106-112 (1990). Much like behavioral audiograms collected by operant conditioning methods (Radizwon et al., J. Comp. Physiol. A. Neuroethol. Sens. Neural Behay. Physiol., 195(10), 961-969 (2009)), the prepulse was varied in intensity and frequency to differentially modulate the startle response. This method has successfully identified behavioral correlates of cochlear damage due to ototoxic drugs (Young J S, Fechter L D, J. Acoust. Soc. Am. 73(5), 1686-1693 (1983)) and temporary threshold shifts due to pure tone acoustic exposure (Walter et al., Open Journal of Acousics 2, 34-49 (2012)).
The inventors have developed a new method of hearing assessment utilizing prepulse inhibition (PPI) of the acoustic startle reflex, a commonly used tool that has been used in many fields of science. The inventors describe using PPI with some modifications to measures detection thresholds in multiple awake animals simultaneously. They found that in control mice PPI audiometric functions are similar to both ABR and traditional operant conditioning audiograms. The hearing thresholds assessed with PPI audiometry in sound exposed mice were also similar to those detected by ABR thresholds one day after exposure. However, three months after exposure PPI threshold shifts were still evident at and near the frequency of exposure whereas ABR thresholds recovered to the pre-exposed level. In contrast, PPI audiometry and ABR wave one amplitudes detected similar losses. PPI audiometry provides a high throughput automated behavioral screening tool of hearing in awake animals. Overall, PPI audiometry and ABR assessments of the auditory system are robust techniques with distinct advantages and limitations, which when combined, can provide ample information about the functionality of the auditory system.
Advantages of this approach for assessing hearing thresholds are numerous. First, the measure is based on a reflex and does not require experience in animal behavior and months of animal training as in other commonly used behavioral paradigms. Second, in contrast to the ABR approach, PPI audiometric functions can be collected in awake animals, avoiding confounds of anesthesia. Finally, a key advantage is that many animals can be tested at once with a short preparation, allowing for high data throughput, and timely data collections at various experimental conditions.
The present invention may be more readily understood by reference to the following figures, wherein:
The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.
Methods of evaluating hearing using pre-pulse inhibition are described. Pre-pulse inhibition is an auditory effect in which an initial pulse of sound can inhibit the startle response that occurs in response to a subsequent loud noise (i.e., auditory startle stimulus). Because the methods rely on evaluation of the startle response, there is no subjective or conscious element to the test, which increases its reliability and ability to be used in subjects that are unable to provide conscious feedback on their response to auditory stimulus.
In one aspect, the present invention provides a method of detecting hearing loss in a subject 10, as shown in
The method includes delivering auditory stimuli to the subject. Auditory stimuli are sound waves capable of being heard by a normal subject. The auditory stimulus can be generated and delivered by an audiometric unit. This can be a simple sound generator by means of which the user is exposed to test signals of different frequencies and sound levels through insert earphones, headphones, free speakers, or bone conduction transducers. The audiometric unit typically includes computer processor programmed/configured to run one or more audiometric test protocols, generating appropriate signals to cause the sound generator to generate one or more sound stimulus in one or both of the subject's ears in accordance with the programmed test protocols. The audiometric test regimen could be a full test designed to generate a complete audiometric profile of the user (for example, testing a series of frequencies across the range of human hearing, such as 20 Hz to 20 kHz for example), or it could be a partial test profile of only a portion of the hearing range (such as 3-6 kHz, for example, which tends to be the range in which damage is most likely to occur from exposure to noisy environments).
The method includes delivering both a pre-pulse auditory stimulus and an auditory startle stimulus to the subject. The auditory startle stimulus is a loud sound sufficient to cause the subject to be startled and exhibit a startle response. Different subjects have differing startle-stimulus response functions, which are known or can be easily determined by one skilled in the art. Suitable startle stimulus typically include one or more audible wavelengths delivered at a volume of from 50 to 130 decibels (dB) sound pressure level (SPL). Lower sound levels are insufficient to stimulate a startle response, while higher sound levels can cause saturation, which decreases the effectiveness of the pre-pulse auditory stimulus. Accordingly, in some embodiments, a startle stimulus from 70 to 110 dB SPL, or from 80 to 100 dB SPL can be used. In some embodiments, the startle stimulus has a loudness of at least 70 dB SPL. Suitable audible wavelengths differ depending on the nature of the subject. For example, suitable audible wavelengths for a human subject range from 20 Hz to 20 kHz, while suitable audible wavelengths for a mouse subject range from 1 kHz to 70 KHz. Typically the startle stimulus will include multiple (e.g., wide-band) audible frequencies. The duration of the start stimulus can be from 5 to 100 milliseconds (ms), with some embodiments using a duration ranging 10 to 60 ms, and other embodiments using a duration ranging from 15 to 30 ms.
A pre-pulse auditory stimulus is delivered to the subject prior to delivery of the auditory startle stimulus. Pre-pulse auditory stimulus are administered at a single frequency (i.e., a pure tone), and a lower volume relative to the startle stimulus. The frequency of the pre-pulse auditory stimulus can be any wavelength audible to the subject, but should overlap with the wavelengths used in the startle stimulus. The pre-pulse auditory stimulus can be delivered at a volume from 10 to 80 dB SPL, while in some embodiments it is delivered at 10 to 70 dB SPL, while in further embodiments the pre-pulse auditory stimulus is delivered at 10 to 60 dB SPL. The duration of the pre-pulse stimulus can be from 5 to 100 milliseconds (ms), with some embodiments using a duration ranging from 10 to 60 ms, and other embodiments using a duration ranging from 15 to 30 ms.
In some embodiments, the method is repeated using pre-pulse auditory stimulus at a plurality of different audible frequencies. This allows for frequency-specific changes (e.g., loss) of hearing to be identified. Repeating the method involves carrying out all of the steps of the method, but using a pre-pulse auditory stimulus having a different audible frequency from what was used previously. For example, the method can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times using different audible frequencies.
The method includes allowing a stimulus gap to pass after delivering a pre-pulse auditory stimulus to the subject, and before delivering an auditory startle stimulus to the subject. The stimulus gap is simply a period of time in which no auditory stimulus is delivered to the subject, in order to distinguish the pre-pulse auditory stimulus from the auditory startle stimulus. The gap should be long enough to distinguish the signals, but not so long that the pre-pulse inhibitory effect is lost. For example, the stimulus gap can have a value in the range of 10 ms to 200 ms. In some embodiments, the stimulus gap has a value in the range of 20 ms to 160 ms, while in further embodiments the stimulus gap has a value in the range of 40 ms to 120 ms. In some embodiments, the pre-pulse auditory stimulus can inhibit the startle response even if the gap is absent, so in some embodiments the stimulus gap can have a value in the range of 0 ms to 200 ms.
The method also includes measuring the startle response of the subject, and determining if the startle response of the subject to the auditory startle stimulus has decreased compared with a control value for the startle response. The startle response is a largely unconscious physical defensive response to sudden or threatening stimulus, such as sudden noise or movement. The onset of the startle response is a startle reflex reaction. The startle reflex is a brainstem reflectory reaction (reflex) that serves to protect vulnerable parts, such as the back of the neck (whole-body startle) and the eyes (eyeblink). There are many various reflexes that can occur simultaneously during a startle response. The fastest reflex recorded in human subjects happens within the masseter muscle or jaw muscle. Electromyography showed the latency response or the delay between the stimulus and the response recorded was found to be about 14 milliseconds. The blink of the eye which is the reflex of the orbicularis oculi muscle was found to have a latency of about 20 to 40 milliseconds. Out of larger body parts, the head is quickest in a movement latency in a range from 60 to 120 milliseconds. The neck then moves almost simultaneously with a latency of 75 to 121 milliseconds. Next, the shoulder jerks at 100 to 121 milliseconds along with the arms at 125 to 195 milliseconds. Lastly the legs respond with a latency of 145 to 395 milliseconds. This type of cascading response correlates to how the synapses travel from the brain and down the spinal cord to activate each motor neuron. Corresponding reflex reactions also occur in other types of subject, such as test animals.
The startle response can be measured in a variety of different ways. For example, when evaluating the start response in human subjects, a preferred measure of the startle response is detection of an eye blink. For evaluating test animals, the inventors used high-speed video recordings to visualize animal startles in order to identify a stereotyped waveform associated with a startle. Based on this information, an algorithm which automatically separates data into either startles or non-startle-related movement was developed. Accordingly, in some embodiments, a startle algorithm is used to measure the startle response of the subject. Mathematical analysis can also be used to normalize startle response magnitudes of individual animals to their body mass. For example, when evaluating the start response of test animals, a mathematical extrapolation is used to convert startle data (force) into center of mass displacement (height). Startle magnitudes measured in force (Newton) were converted to center of mass displacement (COMD; mm). Force was divided by body mass to yield instantaneous acceleration of the COM. The acceleration vector was then integrated to obtain instantaneous velocity and then integrated again to obtain the instantaneous position of the COM. The mathematical conversion normalizes for mass, allowing comparison between animals of different mass, and converts the forces sensed by a piezoelectric startle plate.
The method of detecting hearing loss further includes repeating the steps of delivering the pre-pulse auditory stimulus, allowing a gap of time to pass, delivering an auditory startle stimulus, and measuring the startle response, until the pre-pulse auditory stimulus delivered in that iteration of the method has a decibel value sufficient to cause a decrease in the startle response of the subject. A decrease can be identified by comparing the startle response with a control value for the startle response. A control value can be a startle response previously determined for the subject, or a typical startle response obtained by determining the average startle response from a number of subjects of the same species. When repeating the steps, the pre-pulse auditory stimulus should begin with a low decibel value, which increases each time the steps are carried out, until the pre-pulse auditory stimulus decreases the startle response of the subject as a result of pre-pulse inhibition. The decibel value can be increased in any manner when repeating the steps of the invention. For example, the decibel value can increase by 2, 5, or 10 decibels per iteration of the steps of the method. The decibel level of the pre-pulse auditory stimulus can start, for example, at 40 decibels, followed by pre-pulse auditory stimuli at 45 db, 50 db, 55 db, 60 db, and so forth, until a decrease in the startle response is detected.
Once a decrease in the startle response has been detected, one can determine whether or not the subject has suffered hearing loss. A subject can be determined to have hearing loss if the pre-pulse auditory stimulus required to decrease the startle response is higher than a pre-pulse auditory stimulus threshold value for the subject. Methods of determining a pre-pulse auditory stimulus threshold value for a subject are described herein. In embodiments where pre-pulse auditory stimuli having a variety of different frequencies are used, the method can also be used to characterize the specific frequency or frequencies of the hearing loss.
In some embodiments, the degree of hearing loss is determined. The degree of hearing loss correlates with the degree of difference between the pre-pulse auditory stimulus that caused a decrease in the startle response, and the pre-pulse auditory stimulus threshold value for the subject. Hearing loss can be classified by characterizing the hearing loss in different grades (e.g. “slight”, “moderate” or “severe”) in different frequency ranges (e.g. low-tone range, medium tone range, high-tone range). A specific hearing-loss class can thus be determined by a moderate loss of hearing in the low-tone range, a slight hearing loss in the medium frequency range and a severe hearing loss in the high-tone range. Other differentiating features of different hearing-loss classes are however also possible. For example the class of hearing loss can be characterized based on susceptibility to different forms of treatment; e.g., hearing loss that can be suitably treated only by a hearing aid worn behind the ear with a customized ear adapter piece, or the hearing-loss class a characteristic of which is that an open treatment to compensate for the hearing loss is also suitable.
In some embodiments, the data collected carrying out a method of the invention is statistically transformed. A square root transform was found to be the best at generating a normal distribution of startle responses within each trial type, as assessed using the Anderson-Darling test. This standard statistical transform reduces skew by enhancing the lesser and reducing the larger startle magnitudes. See
In some embodiments, a visual display by means of which the result of the hearing test is shown to the subject and/or the test giver can also be used. The display can provide numeric or qualitative values. For example, the illumination of a red light can be used to indicate that the subject has severe hearing loss. If, on the other hand, an orange-colored light illuminates, this indicates that the subject has moderate hearing loss. A yellow-colored light can indicate that the subject has slight hearing loss, while a green light can, for example, indicate that there is no significant hearing loss present and no hearing aid is necessary. Naturally, there are many other possibilities in addition to the indications already mentioned to indicate the results of hearing tests by visual (e.g. by means of a display), acoustic (e.g. by means of voice output) or tactile means.
A subject, as defined herein, is an animal, preferably a mammal such as a domesticated farm animal (e.g., cow, horse, pig), a pet (e.g., dog, cat), or a non-human test animal (e.g., a rat, mouse, or primate). More preferably, the subject is a human (e.g., a human infant).
In some embodiments, the subject being evaluated is a subject that has been identified as having an increased risk of suffering from hearing loss. Hearing loss is mainly caused by damage to the cochlear hair cells, the sensory cells in the cochlea. Cochlear hair cell damage can result from a number of causes, including age-related damage or loss (presbycusis), acute or chronic noise exposure, acute or chronic drug exposure, e.g., aminoglycoside antibiotics and anti-cancer therapeutics, infections, syndromic and non-syndromic genetic mutations, and autoimmune disease.
In some embodiments, the method of detecting hearing loss can be used to identify and/or guide the treatment of subjects having hearing loss. Treatments for hearing loss include electronic cochlear implants, hearing assistive technology, audiologic rehabilitation, the use of hearing aids, and small molecule compounds for treating hearing loss. See U.S. Patent Publication No. 2015/126507.
In some embodiments, a plurality of subjects are evaluated. In this case, the method of the invention is carried out as described on more than one subject. This can be more readily done because of the relative speed of the method, and the fact that subjects can be evaluated without the need for a conscious response by the subject. For example, the method can be used to evaluate the hearing of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more than 100 subjects. In further embodiments, the method is used to evaluate a plurality of subjects at the same time; i.e., simultaneously.
The ability to evaluate a plurality of subjects simultaneously, without requiring a conscious response by the subject, also renders the invention well suited for screening subjects such as test animals that have been exposed to a drug. Accordingly, in some embodiments, the method further comprises administering a test drug to the subject before delivering a pre-pulse auditory stimulus to the subject. The test drug can be any drug which needs to be tested for having an effect (such as an undesired side-effect) on a subject's hearing.
Another aspect of the present invention is shown in
The pre-pulse auditory stimulus, the stimulus gap, the auditory startle stimulus, and the startle response have the characteristics described herein. Accordingly, steps 32, 34, 36, and 38 of
In some embodiments, the method is used to determine a plurality of pre-pulse auditory stimulus threshold values for a plurality of subjects, which in further embodiments the plurality of pre-pulse auditory stimulus threshold values are determined simultaneously. The methods can be used to determine the pre-pulse auditory stimulus threshold in subjects that cannot give a conscious response, such as a non-human test animal or a human infant.
Another aspect of the present invention is shown in
The method of determining the effect of a drug on hearing includes first determining a pre-pulse auditory stimulus threshold value for the subject. This includes the steps of delivering a pre-pulse auditory stimulus to the subject 52, allowing a stimulus gap to pass 54, delivering an auditory startle stimulus to the subject 56, measuring the startle response of the subject 58, and identifying a pre-pulse auditory stimulus threshold value for the subject when the measured startle response is lower than a control value for the startle response 60.
Once the pre-pulse auditory stimulus threshold value has been determined for a normal subject, a drug is then administered to the subject 62. The drug can be administered to the subject using routine methods such as oral or intravenous administration, and can be administered together with a pharmaceutically acceptable carrier. In some embodiments, the drug is being screened to determine if it might be useful for treating hearing loss, while in other embodiments the drug is being screened to determine if it has an adverse effect on hearing. Examples of compounds that can cause hearing loss include some aminoglycoside antibiotics and anti-cancer therapeutics.
After the drug has been administered, the remaining steps of the method are carried out to determine the effect of the drug on hearing of the subject. These steps include delivering a pre-pulse auditory stimulus to the subject 64, allowing a stimulus gap to pass 66, delivering an auditory startle stimulus to the subject 68, measuring the startle response of the subject, and determining if the startle response of the subject to the auditory startle stimulus has decreased compared with a control value for the startle response 70, repeating steps 64 to 70 until the pre-pulse auditory stimulus administered has a value sufficient to cause a decrease in the startle response of the subject, and determining that the drug has caused hearing loss in the subject if the pre-pulse auditory stimulus required to decrease the startle response is higher than the pre-pulse auditory stimulus threshold value for the subject or determining that the drug has caused hearing improvement in the subject if the pre-pulse auditory stimulus required to decrease the startle response is lower than the pre-pulse auditory stimulus threshold value for the subject 74.
In some embodiments, the method is used to determine a plurality of pre-pulse auditory stimulus threshold values for a plurality of subjects. In other embodiments, the subject is a non-human test animal. In further embodiments, the method is repeated using pre-pulse auditory stimulus at a plurality of different audible frequencies.
An example has been included to more clearly describe a particular embodiment of the invention and its associated cost and operational advantages. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein.
Although PPI audiometry has several advantages over other currently used hearing assessment methodologies, several important questions need to be explored before it can be widely applied. First, it is still unknown whether it is sensitive enough to assess hearing in individual animals, which would be much more beneficial than group averages. Second, the extent of PPI threshold reliability from day to day is also unknown. Third, it is important to know whether PPI audiometry can be used to detect permanent threshold shifts caused by the most common hearing insult, noise-induced hearing loss. The goal of this study was to address these questions and compare PPI audiometry with the traditional ABR approach.
Animals
A total of 16 male CBA/CaJ mice obtained from Jackson Laboratories were used. To avoid startle variability which is known to result from hormone fluctuations of the estrous cycle, female mice were not used in this study. Plappert et al., Physiology & Behavior 84: 585-594 (2005). This phenomenon has also been shown in human subjects (Kumari et al., Schizophrenia Research, 69: 219-235 (2004)). Mice were 12 weeks old at the beginning of the experimental procedures. They were housed in pairs within a colony room with a 12-h light-dark cycle at 25° C.
Ten mice were sound exposed as described below while six unexposed mice were used as controls. The exposed mice were tested during a 3 month period to detect permanent threshold shifts. The 6 control mice were used to test for the consistency of PPI measurements across time.
Acoustic Trauma
Mice were anesthetized with an intramuscular injection of a ketamine/xylazine mixture (100/10 mg/kg). An additional injection (50% of the initial dose) was given 30 min after the initial injection. Mice were unilaterally exposed to a one octave narrow-band noise centered at 12.5 kHz (˜8-17 kHz). This noise was generated using a waveform generator (Tektronix AFG 3021B), amplified (QSC RMX 2450) to 116 dB SPL, and played through a speaker (Fostex T925A Horn Tweeter). The output of the loudspeaker was calibrated with a 0.25-in. microphone (Brüel and Kjaer 4135) attached to a measuring amplifier (Brilel and Kjaer 2525) and found to be ±4 dB between 4 and 60 kHz. During exposure the speaker was located ˜5 cm from the animal's right ear. The left external ear canal was obstructed with a cotton plug and a Kwik-Sil silicone elastomer plug (World Precision Instruments), a manipulation which typically reduces sound levels by 30 to 50 dB SPL. Turner et al., Behavioral Neuroscience 120, 188-195 (2006).
Auditory Brainstem Response Testing
Mice were anesthetized with ketamine/xylazine as during the acoustic trauma. Sterile, stainless-steel recording electrodes (connected to a Tucker Davis Technologies (TDT) RA4LI Low Impedance Headstage) were placed subdermally, one behind each pinna with the reference electrode along the vertex. Tone bursts at 4, 12.5, 16, 20, 25, and 31.5 kHz were presented at increasing sound intensities ranging from 10 to 80 dB SPL in 10 dB steps. Tones were 5 ms duration, 0.5 ms rise/fall time and delivered at the rate of 50/s. ABRs were averaged over 300 repetitions. These waveforms were amplified (TDT RA4PA Medusa Preamplifier), digitized (TDT RZ6 Multi-I/O Processor), and analyzed offline using custom made software (TDT OpenEx). Thresholds, the smallest sound amplitude that evoked a visible ABR, were determined by visually examining the ABR waveforms in response to every sound frequency presented at different sound levels. ABR wave one amplitudes (μV peak to peak) were measured at each intensity/frequency combination for all exposed mice at all time points tested (prior to exposure (control), one day after exposure, and three months after exposure).
Acoustic Startle Hardware/Software
The equipment used to collect all acoustic startle data has been described in detail previously. Longenecker R J, Galazyuk A V, Brain Research 1485, 54-62 (2012). Commercial hardware/software equipment from Kinder Scientific, Inc. was used in behavioral experiments. Each behavioral testing station was lined with anechoic foam to prevent sound reflection and wave cancelling sound echoes (Sonex foam from Pinta Acoustics). A small customization of the hardware's startle stimulus system was made by adding SLA-4 (ART) amplifiers to adjust sound levels to correct for variations in speaker loudness between testing station. Mice restrainers were open walled to allow for maximum sound penetration. Background sound levels within each testing chamber were calibrated with a 0.25-in. microphone (Brüel and Kjaer 4135) attached to a measuring amplifier (Brüel and Kjaer 2525) and found to be less than 40 dB SPL between 4 and 60 kHz. Startle waveforms were recorded using load cell platforms which measure actual force changes during an animal's jump. Each load cell was calibrated with a 100 g weight which corresponds to 1 newton of force. Offline waveform analysis converted these forces into center of mass displacement (in mm). Grimsley et al., J Neurosci Methods 253:206-217 (2015).
Reflex Modification Audiometry: Prepulse Detection Testing
Testing sessions contained two types of stimulus. First, a startle stimulus (wide-band noise, 100 dB SPL, 20 ms in duration, 1 ms rise/fall) was presented alone; this is referred to in the text as startle only (SO) (
Reflex Modification Audiometry: Startle Waveform Identification and Measure
All waveforms collected during testing sessions were analyzed offline using a recently developed automatic method of startle waveform identification via a template matching paradigm. Grimsley et al., J Neurosci Methods 253:206-217 (2015). In this recent study we used high-speed video recordings (1,000 frames/s) to visualize animal startles in order to identify stereotyped waveforms associated with a startle. This allowed us to develop custom software which automatically separates data into either startles or non-startle-related movements. Based on this separation, we have only included trials that resulted in successful startle responses in our data analysis. We also used a mathematical approach to normalize startle response magnitudes of individual animals to their body mass. This mathematical conversion normalizes for mass, allowing legitimate comparisons between animals of different mass.
Reflex Modification Audiometry: Prepulse Detection Threshold Identification
In each session, the magnitudes of the SOs were compared to the magnitudes of startles preceded by prepulses with various frequency and intensity (
Statistical Analyses
Both ABR threshold data and PPI thresholds were evaluated with a repeated measures design because the same animals were used in each condition. Different frequencies (six) and conditions (three) were used as independent variables in the repeated measures analysis of variance (ANOVA). Planned comparisons between specific frequency/intensity combinations were assessed with Fisher's Least Significant Difference (LSD) post-hoc tests. Linear regressions were used to evaluate correlations between PPI and ABR data. Values throughout the manuscript are specified as means or medians±standard error of the mean (SEM). We used an alpha level of 0.05 for all statistical tests. P values significant at the 0.05 level are indicated by one symbol while those significant at the 0.001 level or lower are indicated by two symbols. Due to the complexity of some figures, various symbols have been used to represent significant differences: *=significant difference between the control condition and one day after exposure; #=significant difference between the control condition and three months after exposure; @=significant difference between one day after exposure and three months after exposure.
Results
Consistency of PPI Audiometry
Accuracy and precision are crucial for any methodology that is assessing hearing loss. As such, we tested whether thresholds identified with PPI audiometry were comparable to thresholds determined by behavioral audiograms. Radziwon et al., J. Comp. Physiol. A. Neuroethol. Sens. Neural Behay. Physiol. 195(10), 961-969 (2009). We also assessed both the variation between mice as well as the variation within individual mice across multiple testing sessions. On three consecutive days of testing, thresholds at all frequencies tested (4, 12.5, 20, 25, 31.5 kHz) were below 40 dB SPL with inter-animal differences of usually less than 10 dB (
Comparing PPI and ABR Audiometric Thresholds for Hearing Assessment
To determine if PPI audiometry is sensitive to both temporary and permanent threshold shifts we exposed 10 mice to a one octave narrowband noise centered at 12.5 kHz and presented at 116 dB SPL for 1 hour. Immediately after exposure, PPI thresholds were dramatically increased which resulted from a “flatter” cubic spline function in comparison to the “steep” monotonic function in the control condition, thus contributing to increased detection thresholds (
The effect of noise-induced trauma on hearing was evaluated with PPI and ABR thresholds collected before, one day, and three months after exposure. A repeated measures ANOVA found that both PPI and ABR thresholds were significantly elevated one day (PPI: F(1,53)=75.8, p<0.001; ABR: F(1,53)=18.3, p<0.001) and three months following exposure (
The relationship between PPI and ABR thresholds was directly compared with a linear regression for each condition. In the control condition (pre-exposure) PPI and ABR thresholds had a significant weakly positive correlation (R2=0.3 (F(1,57)=24.4, p<0.001)) (
Comparing PPI and ABR Amplitudes for Hearing Assessment
Recent studies have promoted the importance of examining ABR amplitudes instead of thresholds when evaluating noise induced hearing loss because of the strong link to the loss of suprathreshold auditory nerve fibers which are the most vulnerable during acoustic trauma. Kujawa S G, Liberman M C, J Neurosci 29, 14077-14085 (2009). For this reason we compared prepulse modulated startle magnitudes and the resulting inhibition (PPI) with wave one ABR amplitudes (
Interestingly, when PPI and ABR wave one amplitude deficits were plotted across frequencies as a function of difference from control amplitudes, unique patterns of damage were observed (PPI FIG. 8A2-F2, ABR wave one amplitudes FIG. 8A3-F3, compared in
The relationship between PPI and ABR wave one amplitudes was directly compared with a linear regression for each condition. A very similar pattern was seen between PPI/ABR amplitude correlations (
Effects of Sound Exposure on Startle Magnitude and Probability
The magnitude of the startle response is often decreased either after sound exposure (Longenecker et al., SpringerPlus 3, 542 (2014); Lobarinas et al., Hearing Research 295, 150-160 (2013)) or due to habituation. Both may confound experimental findings by causing a “floor effect”. To test for this possibility, startle-only input/output functions were collected before, one day after, and three months after sound exposure for the group of ten exposed mice (
To further ensure that startle responses are properly assessed, our automatic startle waveform identification software provided us with an assurance that only true startle responses were included in our PPI data analysis (Grimsley et al., J Neurosci Methods 253:206-217 (2015)). This technology is especially important for separating small startle waveforms from non-stimulus-related movements. The percentage of non-startles is stable across the session. The startle probability is directly correlated with the startle magnitude, so if the startle magnitude is stable (lack of habituation), then the startle probability will also remain constant. As mentioned above, each stimulus (prepulse level or startle only) was presented 39 times per session. We found that the startle response magnitude changes that occur within input/output functions (
Habituation to continually presented startle stimulus is also a concern during extended testing paradigms. To ensure habituation was not a significant confounding factor for the ASR in PPI, we monitored the startle magnitudes throughout the testing sessions. A Spearman correlation between startle magnitude and time of testing was run to identify whether magnitudes decreased during testing. The correlation was very small but significant (rs=0.146, p<0.001), suggesting that the startle response magnitudes were actually slightly elevated during recording sessions. This data is supported by previous findings that many strains of mice do not habituate to the acoustic startle reflex. Bullock et al., Behavioral Neuroscience 111 (6): 1353-1360 (1997). This has also been shown specifically for CBA/CaJ mice.
We found that after several refinements, PPI audiometry was capable of measuring pre-pulse detection thresholds in mice much faster (90 minutes vs 9240 minutes) than previously shown in rats. Fechter et al., J. Acoustic. Soc. Am. 84(1), 179-185 (1988). It requires roughly as much time as standard ABR measurements, but can be conducted on multiple animals simultaneously without any anesthesia. Spectrally broad temporary threshold shifts, resulting from sound exposure, can be detected by PPI audiometry and are similar to those detected by ABRs (
What do ABRs and PPI Audiometry Measure?
The auditory brainstem response is by far the most popular methodology to assess the auditory system, as it can rapidly evaluate the functional connectivity of the auditory pathway. These recordings are able to identify gross malfunctions of the auditory system and are a useful tool for assessing the efficacy of sound trauma. The ABR is a non-invasive evoked potential usually conducted in anesthetized animals. It consists of five positive waves, each representing a synchronized neural output generated by auditory structures at and below the level of the inferior colliculus. Melcher J R, Kiang N Y S, Hearing Research 93, 52-71 (1996). This method allows for an in depth frequency specific synopsis of the cochlea and how it changes as a result of acoustic insults. A major advantage of this technique is that it can be conducted on humans, which allows comparison to animal models. Additionally, it can be done non-invasively and rather quickly, which allows screening of multiple animals in a short period of time. However, perhaps the biggest disadvantage of ABRs is the need for anesthesia in animal models which can alter the neural output in confounding ways. Additionally, ABRs can become more difficult to collect in older/larger animals due to increased fat to muscle ratios. Lastly, while it is known that ABRs can assess neural plastic changes that occur in the auditory brainstem, it is less certain if they directly measure neural computations above the level of the inferior colliculus. Sensorimotor gating experiments utilizing PPI of the acoustic startle reflex can also monitor neural plastic changes in these lower auditory structures but importantly, also have the ability to monitor cortical structures which are critically involved in modulating prepulse inhibition. Du et al., J Neurosci 31(38), 13644-13653 (2011). Thus, ABR audiometric functions provide some advantages, but may offer a more complete picture of the auditory system function if combined with a behavioral measure like PPI audiometry.
PPI audiometry is effective because the acoustic startle reflex (ASR) is greatly influenced by preceding stimulus. Hoffman H S, Searle J L. J Comp Physiol Psychol., 60(1), 53-58 (1965); Willott J F, Carlson S, Behavioral Neuroscience 109(3), 396-403 (1995). We have confirmed what was previously found, that prepulses presented at threshold levels can be used to assess an animal's hearing by modulating the startle motor output (
The neuronal circuitry underlying the ASR is straightforward and has been studied extensively. Koch M, Prog Neurobiol 59(2), 107-28 (1999). The primary startle circuit includes the serial connections between the auditory nerve fibers, cochlear root neurons (in some species), and the nucleus reticularis pontis caudalis (PnC) region. The motor output is then sent to the interneurons of the spinal cord resulting in a startle. The simple relationship between evoked stimulus amplitude and the associated startle response magnitude (
When prepulses are added to the ASR construct, the neural circuitry is far more complex (Swerdlow et al., Psychopharmacology 156, 194-215 (2001)). The amount of inhibition that a prepulse has on the startle results from complicated neural computations that arise from various brain regions. The primary circuitry for mediating a prepulse's effect on a startle resides below the level of the colliculi. These nuclei send GABAergic projections that terminate at the nucleus reticularis pontis caudalis. Although this simple circuitry is directly responsible for generating the inhibitory effects on the startle, many higher nuclei can modulate these effects as well. Prepulse modulatory mechanisms are far more complicated and depend on behaviorally salient factors such as inter-stimulus interval, frequency, stimulus duration, prior auditory experience, and habituation. It has been shown that a large degree of top-down modulation can influence prepulses. Larrauri J, Schmajuk N, EXS, 98, 245-278 (2006). The amygdala, hippocampus, prefrontal cortex, auditory cortex and many other structures have been flagged as the source of this top-down modulation. To this extent, it seems that PPI audiometry has the potential to assess large portions of the auditory neuraxis and other neocortical regions. Therefore it is likely that PPI audiometry could be a useful tool to assess neuroplastic changes in awake behaviorally responsive animals. PPI audiometry would be especially suited for labs that already employ the ASR for other purposes such as tinnitus detection (Turner et al., Behavioral Neuroscience 120, 188-195 (2006)) or psychopharmaceutical studies. Phillips et al., J Psycopharmacol; 14(1):40-5 (2000). It should be noted that PPI tests are performed on multiple animals concurrently with no prerequisite training for the animals or the handlers. In this study we found that we could assess auditory thresholds at 6 different frequencies in less than two hours, and future work will aim to reduce this length of time even further. This rate of testing would be exceptionally valuable for drug testing which requires large sample sizes.
Comparison of PPI Audiometry to ABR Assessments
Comparing PPI and ABR Thresholds
The ASR has been shown to be greatly influenced by preceding stimulus and can be used for hearing assessment in laboratory animals and humans. In animals PPI can provide a behaviorally assessed, high throughput solution to assess auditory thresholds in normal hearing and sound exposed animals (
Comparing Startle Modulated Prepulse Inhibition with ABR Wave One Amplitudes
It has been shown that suprathreshold prepulse modulation of the startle reflex can be diminished in mice with age-related hearing dysfunctions. Willott et al., Behavioral Neuroscience, 108, 703-713 (1994). This reduction was also demonstrated in the present study with prepulse modulated startle responses and associated inhibition after sound exposure (FIG. 8A1-F1). In agreement with previous studies (Kujawa S G, Liberman M C, J Neurosci 29, 14077-14085 (2009)), ABR amplitudes in exposed mice decreased over time (FIG. 8A2-F2). The most important finding in this study was that the magnitude of these suprathreshold deficits were frequency specific when measured with both PPI audiometry and ABR wave one amplitudes (
The PPI Model
Considerations and Limitations of PPI Threshold Assessments
Several important factors must be considered when using the cubic spline function to assess auditory thresholds via PPI functions (
A Behavioral Representation of High Threshold ANF Depletion Following Sound Exposure
Recent studies have discovered that while clinically normal threshold levels may be maintained following sound exposure, both mice (100 dB SPL 2 hours; Kujawa S G & Liberman M C, J Neurosci 29, 14077-14085 (2009)) and guinea pigs (106 or 109 dB SPL 2 hours; Lin et al., J Assoc Res Otolaryngol., 12(5):605-16 (2011)) display clear suprathreshold ABR wave one amplitude deficits associated with ANF degeneration. Similarly our exposed mice demonstrate reduced ABR wave one amplitudes (FIG. 8A3-F3). Our ABR wave one amplitudes at intense, suprathreshold sound levels were depressed further than at lower sound levels, which might be expected from an intense sound exposure (116 dB SPL 1 hour). The hidden hearing loss model proposed by Schaette and McAlpine suggests that high threshold low spontaneous rate auditory nerve fibers are preferentially damaged after exposure, leaving the majority of low threshold fibers intact (
Importantly, our PPI methodology was able to test this hypothesis behaviorally. Both the frequency specific ANF damage as well as a general decrease in startle magnitude can be we explained by this model (
Can PPI audiometry explain possible perceptual deficits after high intensity sound exposure? Above we discussed how decreases in inhibition might be explained by ANF fiber loss, but it is possible that these deficits have central origin. Interestingly, the inhibition curve is increasingly depressed at higher intensity prepulses (FIG. 8A2-F2), but not closely following the ABR wave one amplitude function (FIG. 8A3-F3). This implies that the behavioral significance of high threshold auditory nerve fibers might be more severe than previously thought. Clearly the direct loss of these fibers alone, which degenerate from the time of exposure until three months after exposure (as evidenced by changes in inhibition from one day after exposure compared to three months in FIG. 8A2-F2), cannot fully explain the intensity coding issues or dynamic range issues that lead to some of the perceptual PPI deficits shown here. Any number of additional plastic changes in the auditory system following ANF degeneration could lead to these extreme inhibition deficits at high intensity prepulses. Even after unilateral sound exposure it is possible that maladaptive plasticity could lead to bilateral changes throughout the auditory system. Popescu M V, Polley D B, Neuron 65, 718-731 (2010). To our knowledge this is one of the first examples of behavioral evidence of hidden hearing loss in an animal model. Mehraei et al., J Neurosci., 36(13): 3755-3764 (2016). Since a modified version of this PPI audiometric method has already been conducted on humans, it might be possible to determine if these effects in mice are also observed in older adults or sound exposed populations. This would be expected based on recent studies showing that ANFs degenerate as a function of age in all humans. Viana et al., Hearing Research 327: 78-88 (2015).
The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/375,557, filed Aug. 16, 2016, the disclosure of which is incorporated by reference herein.
The present invention was made with Government support under Grant Nos. R01 DC011330 and 1F31 DC013498-01A1 awarded by the National Institute on Deafness and Other Communication Disorders. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62375557 | Aug 2016 | US |
