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.

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

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Neuroscience

1.6K ビュー.  University of Pittsburgh. 瞳孔測定は、複雑な音認識閾値を推定するために使用でき、他の方法と組み合わせて、それらの閾値に対する様々な形態の難聴の影響を決定することができる。この手法は、ヒトおよび訓練を受けていない動物からの同じモダリティのデータを特徴付けるために使用することができる。セットアップは比較的簡単で、侵襲性は最小限です。行動と知覚の尺度は、難?...

ビデオ: 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...

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Dunkin-Hartley guinea pigs are an established animal model for osteoarthritis research. Such studies may benefit from intra-articular injections for various reasons, including investigating novel agents or treating disease. We describe a methodology for intra-articular knee injections in Guinea pigs and subsequent micro-computed tomography analysis assessing arthritis-associated knee changes.

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

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Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

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

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

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

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...