SPS was supported by the University of Sydney postgraduate research scholarship.

All experimental protocols described were approved by the University of Sydney Animal Ethics Committee (approved protocol number: 2018/1308).
1. Animals
NOTE: Mice were obtained from the Australian Rodent Centre (ARC; Perth, Australia) and held at the Medical Foundation Building Animal Facility at the University of Sydney.
<ol>
	<li>Maintain the mice on a normal 12 h light/dark cycle with environmental enrichment.</li>
	<li>Use male and female C57BL/6 mice (3&#8211;5 weeks old) for all experiments.</li>
</ol>
2. Preparation of Solutions
<ol>
	<li>Prepare 1 L of artificial cerebrospinal fluid (ACSF) composed of 29 mM NaHCO3, 11 mM glucose, 120 mM NaCl, 3.3 mM KCl, 1.4 mM NaH2PO4, 2.2 mM MgCl2, 2.77 mM CaCl2.</li>
	<li>Prepare 200 mL of sucrose-ACSF (sACSF) containing 29 mM NaHCO3, 11 mM glucose, 241.5 mM sucrose, 3.3 mM KCl, 1.4 mM NaH2PO4, 2.2 mM MgCl2, 2.77 mM CaCl2. Prior to the inclusion of CaCl2 to the ACSF and sACSF, gas the solutions with carbogen (95 % O2 and 5 % CO2) to establish a pH of 7.4 and avoid calcium precipitation (cloudiness).</li>
	<li>Prepare K+-based intracellular solution composed of 70 mM potassium gluconate, 70 mM KCl, 2 mM NaCl, 10 mM HEPES, 4 mM EGTA, 4 mM Mg2-ATP, 0.3 mM Na3-GTP; with a final pH of 7.3 (adjusted using KOH). 
	NOTE: It is recommended to filter intracellular solutions with 0.22 &#181;m filters and store 0.5 mL aliquots of the solution at -20 &#176;C.</li>
</ol>
3. Preparation of the Brainstem
<ol>
	<li>Prior to brainstem extraction, equilibrate the sACSF with carbogen and cool at -80 &#176;C for 25 min so that an ice slurry is formed.</li>
	<li>Anaesthetize the mouse with isoflurane (3&#8211;5 %) saturated in oxygen (3 mL/min). Once the hind paw reflexes are absent, decapitate the mouse with sharp stainless-steel scissors.</li>
	<li>Expose the skull by making a sagittal incision in the skin using a razor blade (#22 rounded).</li>
	<li>Using the pointed end of a pair of standard pattern scissors make a small incision at the lambda and cut along the longitudinal fissure.</li>
	<li>Carefully reflect away the paired parietal bones and the occipital bones using a pair of shallow-bend Pearson rongeurs. 
	NOTE: During this whole procedure the brain is continuously bathed in situ using the previously prepared ice-cold sACSF slurry.</li>
	<li>Isolate the brainstem from the forebrain and its bony encasing using a razor blade (#11 straight) to cut down the parieto-occipital sulcus and at the caudal medulla.</li>
	<li>Mount the isolated brainstem ventral end down on a previously cut trapezoidal polystyrene block. Remove excess fluid around the dissected tissue with a wick of tissue paper to ensure good tissue adhesion to the cutting stage. 
	NOTE: The polystyrene block is cut in a trapezoidal shape, to ensure the rostral end of the midbrain fits and tapers into the spinal cord.</li>
	<li>Use cyanoacrylate glue to fix the polystyrene block with the attached brainstem rostral end down to the cutting stage.</li>
	<li>Using an advance speed of 0.16 mm/s and vibration amplitude of 3.00 mm, prepare 200 &#181;m transverse slices of the MVN. 
	NOTE: Location of the MVN is determined using the Paxinos and Franklin mouse brain atlas (Figures 79&#8211;89)20. The MVN (listed as MVe in atlas) lies immediately ventrolateral to the 4th ventricle and is largest right before the attachment of the cerebellum (between the inferior colliculi and the obex).</li>
	<li>Use a plastic-trimmed pipette to transfer slices onto a filter paper disc sitting in carbogenated ACSF at 25 &#176;C for at least 30 min prior to recording.</li>
</ol>
4. Instruments
<ol>
	<li>Use a standard electrophysiological setup to perform whole-cell patch clamp techniques21.</li>
	<li>Prepare micropipettes using a two-step protocol (heat step 1: 70; heat step 2: 45) on a micropipette puller (see the Table of Materials). Micropipettes should have a final resistance ranging 3&#8211;5 M&#8486; with internal solution when placed in the bath. 
	NOTE: Settings used may vary depending on the temperature within room and can change quite frequently.</li>
</ol>
5. Whole-cell Patch Clamp Electrophysiology
<ol>
	<li>To obtain whole-cell patch clamp recordings from individual neurons in the MVN, a K+-based internal solution is used within the recording pipette.</li>
	<li>Transfer a single tissue slice from the incubation chamber to the recording chamber and secure the slice using a nylon thread on a U-shaped weight. Continuously perfuse the recording chamber with carbogenated-ACSF at 25 &#176;C at a flow rate of 3 mL/min.</li>
	<li>After filling a micropipette with internal solution, locate the MVN using a low power (10x) objective lens. Using a high-power (40x) objective, individual neurons within the MVN can be located. 
	NOTE: Cell quality is essential in ensuring quality recordings and durability of the cell when attempting to achieve the whole-cell configuration. A good cell will demonstrate spherical shape, a reflective cell membrane and an invisible nucleus. A bad cell will have a large visible nucleus (egg-like) and a swollen/shrunken appearance.</li>
	<li>Before breaching the tissue with the pipette, apply a small amount of positive pressure to push debris away from the pipette tip.</li>
	<li>Move the pipette using the micromanipulator towards the chosen neuron and a small dimple should form on the neuronal membrane. Release positive pressure and apply a small amount of negative pressure.</li>
	<li>Once a 1 G&#8486; seal is achieved, apply gentle short and sharp positive pressure to the pipette holder through the suction port to rupture the membrane and create a whole-cell configuration.</li>
	<li>Make whole-cell current clamp recordings using standard techniques21,22.</li>
</ol>
6. Applying Sinusoidal and Stochastic Noise to Individual Medial Vestibular Nucleus Neurons
<ol>
	<li>Apply the stochastic and sinusoidal noise at a range of amplitudes from 3 to 24 pA to determine neuronal threshold and firing rate.</li>
	<li>Determine the sensory threshold by grouping lower and higher stimulus intensities and perform an ANOVA to observe any differences (as shown in Supplementary Figure 1).</li>
	<li>Calculate the average firing rate over the 10 s period where the depolarizing current step was/will be injected for each individual current level (i.e., 7 total episodes; Figure 1).</li>
	<li>Use the average firing rate values to generate a firing rate versus current plot and perform a linear regression analysis to determine the gradient of the line of best fit. The gradient of the line of best fit is indicative of the neuronal gain22.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60044/60044fig1.jpg" /> 
Figure 1: Diagrammatic profiles of control, sinusoidal and stochastic noise protocols. (A) Control (no noise) protocols applied to MVN neurons. (B) Sinusoidal noise protocol with a frequency of 2 Hz. (C) Stochastic noise protocols where majority of the power spectrum is &#8804;2 Hz. Each protocol presented here has an amplitude of &#177;6 pA with a 10 s depolarizing current increasing by 10 pA up to 50 pA. The true stimulus does not have a depolarizing current step and is therefore the first episode of these protocols to determine neuronal gain changes. <a href="https://www.jove.com/files/ftp_upload/60044/60044fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

The effects of galvanic vestibular stimulation (GVS) on the vestibular system has been highlighted in vivo in humans3,13,23, guinea pigs14, rodents18 and non-human primates24. However, none of these studies have assessed the direct impact of electrical noise on the sensitivity of individual neurons in the vestibular system. Here we demonstrate the first in v...

The vestibular (or balance) system controls our sense of balance by integrating auditory, proprioceptive, somatosensory and visual information. Degradation of the vestibular system has been shown to occur as a function of age and can result in balance deficits1,2. However, therapies targeting the functioning of the vestibular system are scarce.
Galvanic Vestibular Stimulation (GVS) has been shown to improve balance measures, autonomic functioning and other sensory modalities within humans3,4,5,6. These improvements are said to be due to the Stochastic Resonance (SR) phenomenon, which is the increase in the detection of weaker signals in non-linear systems via the application of subthreshold noise7,8. These studies have shown improvements in static9,10 and dynamic11,12 balance, and vestibular output tests such as Ocular Counter Roll (OCR)13. However, many of these studies have used different combinations of stimulus parameters such as white noise9, colored noise13, different stimulus frequency ranges and thresholding techniques. Therefore, optimal stimulus parameters remain unknown and this protocol can assist with determining the most effective parameters. Besides stimulus parameters, the type of stimulus is also important in therapeutic and experimental efficacy. The above work in humans was performed using electrical noise stimuli, whilst much of the in vivo animal work has used mechanical14,15 or optogenetic16 noise stimuli. This protocol will use electrical noise to examine the effects on vestibular nuclei.
Previously, application of GVS to stimulate primary vestibular afferents was been performed in vivo in squirrel monkeys17, chinchillas18, chicken embryos15 and guinea pigs14. However, only two of these studies examined the effect GVS has on the gain of primary vestibular afferents14,15. These experiments were performed in vivo meaning that the precise patterns of stimulation imposed on vestibular nuclei cannot be determined. To our knowledge, only one other study has applied stochastic noise to individual enzymatically dissociated neurons in the central nervous system19. However, no experiments have been performed in the central vestibular nuclei to assess appropriate stimulus parameters and thresholding techniques, making this protocol more precise in determining stimulus effects on individual neurons within the vestibular nuclei.
Here, we describe how to apply sinusoidal and stochastic (electrical) noise directly to individual neurons in the medial vestibular nucleus (MVN), determine neuronal threshold and measure changes in gain/sensitivity.

<table><tbody><tr><td>CaCl</td><td>Scharlau</td><td>CA01951000</td><td>Used for ACSF and sACSF</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G8270</td><td>Used for ACSF and sACSF</td></tr><tr><td>EGTA</td><td>Sigma</td><td>E0396-25G</td><td>Used for K-based intracellular solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-25G</td><td>Used for K-based intracellular solution</td></tr><tr><td>KCl</td><td>Chem-supply</td><td>PA054-500G</td><td>Used for ACSF, sACSF and intracellular solution</td></tr><tr><td>K-gluconate</td><td>Sigma</td><td>P1847-100G</td><td>Used for K-based intracellular solution</td></tr><tr><td>Mg-ATP</td><td>Sigma</td><td>A9187-500MG</td><td>Used for K-based intracellular solution</td></tr><tr><td>MgCl</td><td>Chem-supply</td><td>MA00360500</td><td>Used for ACSF and sACSF</td></tr><tr><td>Na3-GTP</td><td>Sigma</td><td>G8877-100MG</td><td>Used for K-based intracellular solution</td></tr><tr><td>NaCl</td><td>Chem-supply</td><td>SO02270500</td><td>Use for ACSF and intracellular solution</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Ajax</td><td>AJA471-500G</td><td>Used for ACSF and sACSF</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761-1KG</td><td>Used for ACSF and sACSF</td></tr><tr><td>Sucrose</td><td>Chem-supply</td><td>SA030-500G</td><td>Used for sACSF</td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>1169567762</td><td>Used for anaesthetising mice</td></tr><tr><td>EQUIPMENT</td><td></td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Warner instruments</td><td>GC150T-7.5</td><td>1.5 mm OD, 1.16 mm ID, 7.5 cm length</td></tr><tr><td>Data acquisition software</td><td>Axograph</td><td></td><td>Used for electrophysiology and analysis</td></tr><tr><td>Friedmen-Pearson Rongeurs</td><td>World precision instruments</td><td>14089</td><td>Used for dissection</td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PP-830</td><td>Used for micropipette</td></tr><tr><td>Multiclamp amplifier</td><td>Axon instruments</td><td>700B</td><td>Used for electrophysiology</td></tr><tr><td>pH meter</td><td>Sper scientific</td><td>860033</td><td>Used for internal solution</td></tr><tr><td>Standard pattern scissors</td><td>FST</td><td>14028-10</td><td>Used for dissection</td></tr><tr><td>Sutter micromanipulator</td><td>Sutter</td><td>MP-225/M</td><td>Used for electrophysiology</td></tr><tr><td>Upright microscope</td><td>Olympus</td><td>BX51WI</td><td>Used for electrophysiology</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200</td><td>Used for slicing brain tissue</td></tr></tbody></table>

stochastic noise application for the assessment of medial vestibular nucleus neuron sensitivity in vitro

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed to be due to the stochastic resonance (SR) phenomenon, which is defined as application of a low-level/subthreshold stimulus to a non-linear system to increase detection of weaker signals. However, it is still unknown how SR exhibits its positive effects on human balance. This is one of the first demonstrations of the effects of sinusoidal and stochastic noise on individual neurons. Using whole-cell patch clamp electrophysiology, sinusoidal and stochastic noise can be applied directly to individual neurons in the medial vestibular nucleus (MVN) of C57BL/6 mice. Here we demonstrate how to determine the threshold of MVN neurons in order to ensure the sinusoidal and stochastic stimuli are subthreshold and from this, determine the effects that each type of noise has on MVN neuronal gain. We show that subthreshold sinusoidal and stochastic noise can modulate the sensitivity of individual neurons in the MVN without affecting basal firing rates.

Initial recordings can provide information about the effects that sinusoidal and stochastic noise have on basal firing rates of individual MVN neurons and how the stimuli effect the gain of neurons. Figure 2 shows that neither sinusoidal nor stochastic noise change basal firing rates of MVN neurons when compared to control (no noise) recordings. This information is crucial for determining the threshold of the individual neurons. During the application of galvanic vestibul...

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Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...

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5.1K Views.  University of Sydney. Dit protocol is een van de eerste demonstraties van de directe toepassing van elektrische ruis op individuele neuronen in weefselsegmenten. Om neuronale reacties op elektrische stimuli te observeren. Het belangrijkste voordeel van deze techniek is, dat we in staat zijn om een specifieke set van stimulus parameters op individuele neuronen te beoordelen.Deze techniek maakt het mogelijk voor de optimalisatie van stimulus parameters om bepaalde, functionele neuronale types richten. Dit hee...

Video: Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Behavior

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Estimating Vestibular Perceptual Thresholds Using a Six-Degree-Of-Freedom Motion Platform

Vestibular perceptual thresholds refer to the motion intensity required to enable a participant to detect or discriminate a motion based on vestibular input. Using passive motion profiles provided by six degree-of-motion platforms, vestibular perceptual thresholds can be estimated for any kind of motion and thereby target each of the sub-components of the vestibular end-organ. Assessments of vestibular thresholds are clinically relevant as they complement diagnostic tools such as caloric irrigation, the head impulse test (HIT), or vestibular evoked myogenic potentials (VEMPs), which only provide information on sub-components of the vestibular system, but none of them allow for assessing all components. There are several methods with different advantages and disadvantages for estimating vestibular perceptual thresholds. In this article, we present a protocol using an adaptive staircase algorithm and sinusoidal motion profiles for an efficient estimation procedure. Adaptive staircase algorithms consider the response history to determine the peak velocity of the next stimuli and are the most commonly used algorithms in the vestibular domain. We further discuss the impact of motion frequency on vestibular perceptual thresholds.

In this article, we describe the methods, procedures, and technologies required to estimate vestibular perceptual thresholds using a six-degree-of-freedom motion platform.

Vestibular perceptual thresholds refer to the motion intensity required to enable a participant to detect or discriminate a motion based on vestibular ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Biology

Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called magnetic vestibular stimulation (MVS). In this article, we present an experimental setup in a 7T MRT scanner (MRI scanner) that allows the investigation of the influence of strong magnetic fields on nystagmus as well as perceptual and cognitive responses. The strength of MVS is manipulated by altering the head positions of the participants. The orientation of the participants&#39; semicircular canals with respect to the static magnetic field is assessed by combining a 3D magnetometer and 3D constructive interference in steady-state (3D-CISS) images. This approach allows to account for intra- and inter-individual differences in participants&#8217; responses to MVS. In the future, MVS can be useful for clinical research, for example, in the investigation of compensatory processes in vestibular disorders. Furthermore, it could foster insights into the interplay between vestibular information and cognitive processes in terms of spatial cognition and the emergence of self-motion percepts under conflicting sensory information. In fMRI studies, MVS can elicit a possible confounding effect, especially in tasks influenced by vestibular information or in studies comparing vestibular patients with healthy controls.

In this article, we describe the experimental setup, material, and procedures to assess reflexive eye movements, self-motion perception, and cognitive tasks under magnetic vestibular stimulation, as well as the anatomical orientation of the vestibular organs, in a 7 Tesla Magnetic resonance tomography (7T-MRT) scanner.

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique

Source: Stefani, S. P., et al. <a href="https://app.jove.com/v/60044">Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro.</a> J. Vis. Exp. (2019).
This video demonstrates a protocol involving the application of subthreshold noise currents to medial vestibular nucleus neurons using the whole-cell patch-clamp technique to study the impact of the noise currents on neuronal sensitivity to electrical stimuli.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Animals
NOTE: Mice were obtained ...

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

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