We acknowledge Drs. Jing Bing Xue and Ruth Anne Eatock for their early contributions to these methods. This work was supported by NIH NIDCD R03 DC012652 and NIH NIDCD DC012653S, and R01 DC0155512 to RK and T32 DC009975 to DB, NN, and KR.

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The authors declare that there are no conflicts of interest.

The methods presented here are specific to recordings from isolated neurons; previous studies have focused on recordings from axon terminals in a semi-intact preparation. When compared to existing terminal recording techniques, isolated recordings offer superior space-clamp and iso-potential behavior. In addition, this protocol provides access to a broader sample of neurons, since only calyx-bearing subpopulations are accessible in semi-intact recordings of the vestibular epithelia. Finally, isolated recordings allow for the use of the perforated-patch technique, which prevents disruption of the intracellular milieu that is often interrupted by the dialysis between the intracellular solution and cytosol in ruptured-patch recordings.
Successful recordings rely first on the quality of the isolated and cultured somata. A critical step in cell survival is the force required during trituration to produce the isolated neurons. A gentle hand is vital to cell survival. Trituration pipettes should be placed high in the bubble of L-15 solution to prevent forcefully expelling neurons onto the bottom of the dish. If issues of cell survival arise, extra care should also be taken to prevent the formation of air bubbles or in breaking the bubble of solution, which prevents the dissociated cells from settling onto the coverslip. It is also best to have heat-polished trituration pipettes available in a variety of sizes, so that investigators have the flexibility to choose one with a diameter such that the ganglion experiences mild resistance as it passes through the pipette. Heat-polishing the pipettes reduces damage caused by passing the ganglion over rough edges of glass. Leaving a rough edge may initially appear to be more effective in breaking up the ganglia, but this approach damages the somata and reduces survival after the period in culture. A final piece of advice is to jealously safeguard a successful trituration pipette.
Another factor that is critical to cell survival is the duration of treatment with digesting enzymes. When determining enzyme time, investigators need to carefully adjust the time to ensure that the tissue breaks up well, but not so much as to impact cell survival. We find that the impact of enzymatic treatment on cell survival and ion channel properties is minimal, as data gathered from enzyme-treated cells appear to be consistent with other methods that do not rely on this approach19,26. Although enzymatic treatment minimizes the force required to triturate, it does come with the limitation that enzymatic digestion (particularly based on trypsin alone or papain) is known to cause damage to ion channels27. Thus, although we recommend some guidelines for enzyme timing dependent on the age of the animal, it is best to be prepared to adjust the time based on the outcomes. Finally, we encourage a &#39;less is more&#39; approach to trituration. This means resisting the urge to form a single-cell suspension and stopping trituration after three or four passes, even if there are several large chunks of tissue remaining.
An advantage of culturing is that it appears to clean the somatic cell membrane and promotes shedding of the myelin-forming glial cells, which then allows for more successful patch-clamping from the neurons. Thus, the isolated and cultured ganglion preparation is especially useful for extending patch-clamp recordings past the 1st postnatal week, after which the cell bodies become progressively covered with myelin, impeding the patch-clamp electrode. Ion channel activity observed in patch-clamp recordings from isolated plus short-term cultured (&#60;24 h) VGNs beyond the 2nd postnatal week are consistent with zonal and maturational changes in ion channel expression seen by immunohistochemistry and direct recordings from calyx terminals in vestibular epithelia28,29. However, it should be noted that prolonged culturing aided by neurotrophic factors and antibiotics may also affect ion channels30,31,32,33,34. Any application of these techniques must carefully consider the impact of these factors on the underlying biophysics of the neurons and their ion channels30,31,32,33,34.
Overall, patch-clamp recordings from isolated and cultured somata are suitable for studying diversity in the ion channel properties of inner ear neurons. The long-term stability of the perforated-patch configuration is especially suitable for studying ion channels such as HCN, whose activation properties are subject to modulation via cytosolic second messengers.

The bipolar neurons of the vestibulocochlear nerve connect the sensory hair cells of the inner ear to the brainstem. They are principal carriers of information about sound and head movements; damage to these important cells leads to deafness and balance disorders. The vestibular and auditory portions of the nerve are each comprised of distinct cell types that are&#160;morphologically and functionally diverse1,2. In the vestibular system, two afferent subpopulations fire spontaneously at intervals that are either regular or irregular2. Afferent spike timing is thought to reflect an underlying diversity in ion channel composition3,4. In the auditory system, there are two main subpopulations of spiral ganglion neurons (SGNs); whereas Type I SGNs contact individual inner hair cells5, Type II SGNs contact multiple outer hair cells5. In vitro recordings from semi-intact and organotypic cultures suggest differences in the membrane properties of Type I and Type II SGNs6,7.
Many ion channels and neurotransmitter receptors found at the terminals of these neurons are also found in their cell bodies. As such, cultures of the isolated vestibular and the spiral ganglion somata can be studied in vitro to understand how ion channels and neurotransmitter receptors contribute to the response of these neurons. The compact morphology of the isolated cell bodies allows for high-quality electrical recordings, suitable for detailed characterization of voltage-gated ion channels and neurotransmitter receptors. Easy access to a representative variety of neuron subtypes allows for high-throughput analysis of cell diversity.
This article presents a method for isolating and culturing dissociated ganglion cell bodies from the superior portion of the vestibular ganglion in rats at postnatal day (P)9 to P20. Suggestions are also provided for extending these methods to the spiral ganglion, in addition to the steps required for successfully extracting, dissociating, and plating the ganglion cells. These methods are an evolution of those devised in publications from various laboratories8,9,10. Also included in this paper is guidance for selecting healthy cells for patch-clamp recordings.
Finally, the protocol outlines the procedure for patch-clamp recording using the perforated-patch configuration11. Though the perforated-patch configuration is more time-consuming and more technically challenging than the more common ruptured-patch configuration, it is better for maintaining the cytoplasmic milieu that allows for long and stable recording sessions. The benefits of this recording configuration are illustrated here through the improved stability of hyperpolarization-activated cationic currents in perforated-patch relative to ruptured-patch recordings.
This protocol is organized into five sections. Sections 1-3 describe solutions and tools that can be prepared and stored ahead of time. Section 4 describes the steps for dissecting and plating the vestibular and SGNs. Section 5 describes the steps for recording from the neurons after a period in culture. In our hands, section 4 and section 5 are performed over a period of 2 consecutive days.

<table><tbody><tr><td>Amphotericin</td><td>Sigma-Aldrich</td><td>A4888-100MG</td><td>For perforated patch recordings.</td></tr><tr><td>ATP di-sodium</td><td>Sigma-Aldrich</td><td>A7699</td><td>Additive to internal solution</td></tr><tr><td>B27 Supplement (50x), serum free</td><td>Thermo Fisher Scientific</td><td>17504044</td><td>additive to culture medium, for SGN</td></tr><tr><td>Beakers (1000, 100, 10) milliliter</td><td></td><td></td><td></td></tr><tr><td>bench-top centrifuge</td><td>USA Scientific</td><td>2641-0016</td><td></td></tr><tr><td>Bunsen burner</td><td></td><td></td><td></td></tr><tr><td>CaCl2</td><td>J.T. Baker</td><td>1311-01</td><td>Additive to internal solution</td></tr><tr><td>Collagenase</td><td>Sigma-Aldrich</td><td>C5318</td><td>one out of three enzyme to digest tissue</td></tr><tr><td>Coverglass, rectangular, #1 thickness, 22x40&amp;nbsp;</td><td>Warner Instruments</td><td>64-0707</td><td></td></tr><tr><td>DMSO</td><td>Biotium</td><td>90082</td><td></td></tr><tr><td>Dnase I,from bovine pancreas</td><td>Sigma-Aldrich</td><td>11284932001</td><td>one out of three enzyme to digest tissue</td></tr><tr><td>Dumont #3 Forceps (Blunt)</td><td>Fine Science Tools</td><td>11231-30</td><td></td></tr><tr><td>Dumont #5 Forceps (Fine)</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Dumont #55 Forceps (Fine)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E0396</td><td>Additive to internal solution</td></tr><tr><td>Electrode Puller</td><td>Narashige</td><td>PC-10</td><td></td></tr><tr><td>Epi-illumination light source&amp;nbsp;</td><td>Zeiss&amp;nbsp;</td><td>CL 1500 ECO</td><td></td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>2716</td><td>for cleaning head and around dissection bench</td></tr><tr><td>Filamented Borosilicate Capillaries for electrodes</td><td>Sutter Instruments</td><td>BF140-117-10</td><td></td></tr><tr><td>Fine-edged dissection blade</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Glass Pasteur Pipettes</td><td>VWR</td><td>14673-010</td><td>to pull trituration pipettes</td></tr><tr><td>Heat-inactivated Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>16140063</td><td>additive to culture medium</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-100G</td><td>for pH buffering all solutions in protocol</td></tr><tr><td>Hot plate / magnetic stirrers&amp;nbsp;</td><td>VWR</td><td>76549-914</td><td></td></tr><tr><td>Insulated bucket filled with ice</td><td></td><td></td><td>to keep all samples and solutions cool</td></tr><tr><td>K2SO4, Potassium Sulfate</td><td>Sigma Aldrich</td><td>P9458-250G</td><td>Additive to internal solution</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P93333</td><td>Additive to internal solution</td></tr><tr><td>KOH (1 M)</td><td>Honeywell</td><td>319376-500ML</td><td>To bring internal solution to desired pH.</td></tr><tr><td>Large Spring Scissors</td><td>Fine Science Tools</td><td>14133-13</td><td></td></tr><tr><td>Leibovitz medium&amp;nbsp;</td><td>Sigma Aldrich</td><td>L4386</td><td>dissection and bath solutions&amp;nbsp;</td></tr><tr><td>Low-profile-bath recording chamber for culture dishes</td><td>Warner Instruments</td><td>64-0236</td><td></td></tr><tr><td>luer-lok syringes, 30 ml</td><td>BD</td><td>302832</td><td>for drawing L-15/HEPES/HEPES solution.</td></tr><tr><td>MEM + Glutamax Supplement</td><td>Fisher Scientific</td><td>41-090-101</td><td>base of the culture medium</td></tr><tr><td>MgCl2-Hexahydrate</td><td>Sigma-Aldrich</td><td>M1028</td><td>Additive to internal solution</td></tr><tr><td>microFil needle for filling micropipettes - 34 gauge&amp;nbsp;</td><td>World Precision Instruments</td><td>MF34G</td><td></td></tr><tr><td>Microforge</td><td>Narashige</td><td>MF-90</td><td>For electrode polishing.</td></tr><tr><td>N2 Supplement (100x)</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td>additiive to culture medium, for SGN</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>Additive to internal solution</td></tr><tr><td>NaOH (1 M)</td><td>Thomas Scientific</td><td>319511-500ML</td><td>for titration pH</td></tr><tr><td>Osmometer</td><td>Advanced Instruments Inc.</td><td>3320</td><td></td></tr><tr><td>Oxygen, Medical grade, with adequate regulator and tubing</td><td>USC Material Management</td><td>MEDOX200 (Identifier: 00015)</td><td>for dissolving into dissection and bath solutions</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM992</td><td></td></tr><tr><td>Pasteur pipette bulb (3 ml)</td><td>Fisher Scientific</td><td>03-448-25</td><td>bulb for trituration pipettes</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>additive to prevent contamination of culture medium</td></tr><tr><td>Pentobarbital based euthanasia solution (e.g., Fatal Plus. 50 &amp;ndash; 60 mg/kg dosing)&amp;nbsp;</td><td>MWI Animal Health</td><td>15199</td><td>for euthanasia</td></tr><tr><td>PES membrane filters ,&amp;nbsp; 0.2 micrometer&amp;nbsp;</td><td>Nalgene</td><td>566-0020</td><td>for filtering solutions</td></tr><tr><td>PES membrane sterile syringe filters, 0.22 um, 30 mm&amp;nbsp;</td><td>CELLTREAT</td><td>229747</td><td>for filtering solutions drawn into syringes</td></tr><tr><td>Petri dishes, 35 x 10 mm</td><td>Genessee Scientific</td><td>32-103</td><td>for micro dissection and to hold Tip dip solution in perforated-patch configuration</td></tr><tr><td>Petri Dishes, 60 x 15 mm</td><td>Midland Scientific</td><td>P7455</td><td>for gross dissection</td></tr><tr><td>pH Meter</td><td>Mettler Toledo</td><td>Model S20</td><td></td></tr><tr><td>Pipettors (1000, 200, 10) microliter</td><td>USA Scientific</td><td></td><td></td></tr><tr><td>Poly-d-lysine coated glass bottomed culture dish</td><td>Mattek</td><td>P35GC-0-10-C</td><td>to plate neurons for culture</td></tr><tr><td>Quick change platform, heated base, for 35 mm culture dishes</td><td>Warner Instruments</td><td>64-0375</td><td></td></tr><tr><td>Reference Cell</td><td>World Precision Instruments</td><td>RC1T</td><td></td></tr><tr><td>Scalpel blade</td><td>Miltex</td><td>4-315</td><td></td></tr><tr><td>Scalpel Handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Scientific Scale</td><td>Mettler Toledo</td><td>XS64</td><td></td></tr><tr><td>Serological Pipettes (10, 25) milliliter</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Silicone Grease Kit (for sealing coverglass and chamber)</td><td>Warner Instruments</td><td>64-0378</td><td></td></tr><tr><td>Small Animal Guillotine</td><td>Kent Scientific</td><td>DCAP</td><td></td></tr><tr><td>Small animal guillotine</td><td>Kent Scientific</td><td>DCAP</td><td>for decapitation if dissecting rats older than P15.</td></tr><tr><td>Stereo Dissection Microscope&amp;nbsp;</td><td>Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Straight surgical scissors</td><td>Fine Science Tools</td><td>14060-09</td><td></td></tr><tr><td>Syringe (3, 10, 30) milliliter</td><td></td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma Aldrich</td><td>T1426</td><td>one out of three enzyme to digest tissue</td></tr><tr><td>Tuberculin syringe&amp;nbsp;</td><td>Covidien</td><td>8881500105</td><td>for delivering euthanasia solution by intraperitoneal injection</td></tr><tr><td>Vannas Spring Scissor, 2.5 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Volumetric flask, 1000 milliliter</td><td></td><td></td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>945300</td><td></td></tr><tr><td>Water, sterile u ltrapure, R&gt;18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system)</td><td>Millipore-Sigma</td><td>CDUFBI001</td><td></td></tr></tbody></table>

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.

Running voltage-clamp protocols by applying families of voltage steps reveals the voltage-dependent activation of a variety of different families of currents. Representative examples of whole-cell currents evoked from a VGN and adapted from published recordings13 are shown in Figure 1A,B. Applying depolarizing voltages (Figure 1B) activates an inward current (negative by convention) that activates and inactivates very rapidly (Figure 1A). This is stereotypical for the voltage-gated properties of sodium channels14,15,&#160;which principally drive the upstroke of action potentials16,17. Depolarization also activates a long-lasting and relatively slow-activating outward current that drives the downstroke of an action potential. Pharmacology has revealed that these currents are largely carried by a variety of potassium channels in VGNs3,18,19,20,21.
Deep, long-lasting hyperpolarizing voltages evoke a slow-activating inward current carried by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels22 (Figure 1C). The activation of these currents can be studied using the tail-current protocol shown in Figure 1D. Here, the activation percentage of the current is probed by plotting the current that flows during the tail step (Itail) as a function of the conditioning voltage. The current-voltage activation curve has a sigmoidal shape (Figure 1E). Although this channel is gated by soluble second messengers such as cyclic AMP (cAMP), the activation curves measured using the perforated-patch configuration are stable throughout a long recording. In contrast, the size and voltage-activation range of the channel are altered (presumably due to the washout of cytosolic components) when the recording is made in the ruptured-patch configuration.
Neuronal diversity is illustrated by the range of firing patterns elicited when currents are injected into different VGNs and SGNs (Figure 2; top and bottom, respectively). Some neurons fire only at the onset of current injection, while others fire multiple times. This heterogeneity reflects a fundamental diversity in the composition of underlying sodium and potassium channels in both VGNs and SGNs23,24,25.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64908/64908fig01.jpg" /> 
Figure 1: Examples of voltage-clamp protocols for measuring diverse groups of ionic currents. (A,B) Example whole-cell currents (A) induced by a family of voltage steps (B). The net inward sodium currents (negative currents) are identified here by their transient activation and inactivation (labeled arrow, Na+). The net outward currents (labeled arrow, K+, long-lasting positive currents) are carried largely by potassium ions and have much slower activation and inactivation kinetics than sodium currents. (C,D) HCN currents are activated by a family of long-duration hyperpolarizing voltages. (E,F) Stability of ion channel characterization in the perforated-patch at different time points during recording. The voltage-dependent activation curves of HCN currents were measured in a perforated-patch configuration I. Activation curves of HCN currents during a ruptured-patch configuration (F). Images C-F have been modified from13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64908/64908fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64908/64908fig02.jpg" /> 
Figure 2: Diverse firing patterns are evoked by injecting steps of currents. (A) Firing patterns in five vestibular ganglion somata. (B) Firing patterns in five SGNs. (C) Corresponding current steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64908/64908fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings at JoVE.com

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

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 ...

isolating-culturing-vestibular-spiral-ganglion-somata-from-neonatal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.1K Views.  University of Southern California. Veel ionkanalen en neurotransmitterreceptoren die worden aangetroffen aan de uiteinden van auditieve en vestibulaire afferente neuronen worden ook aangetroffen in hun cellichamen. Culturen van geïsoleerde cellichamen kunnen in vitro worden bestudeerd om te begrijpen hoe deze ionkanalen en receptoren de reacties van de neuronen beïnvloeden. De compacte morfologie van de cellichamen maakt elektrische opnames van hoge kwaliteit mogelijk om de spanningsafhankelijke eigenschappen van de ionkanal...

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

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

Biochemistry

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

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 ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

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.

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 ...

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.remotexs.ntu.edu.sg/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 ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from a Flat-Mount Mouse Retina

Source: Tu, H., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54608">Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina.</a> J. Vis. Exp. (2016).
This video demonstrates a whole-cell patch-clamp technique for electrophysiological recording from a flat-mount mouse retina. A thin glass pipette with an electrode is used to approach starburst amacrine cells (SACs) within the retina. After forming a seal, suction is applied to rupture the membrane and form the whole-cell patch-clamp configuration. Finally, the flow of ions through the SAC synaptic receptors is measured using the configuration.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. External and Internal Solutions

	...

Culturing Murine Spiral Ganglion Neuron Explants on Multielectrode Arrays

Source: Hahnewald, S., et al <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54538">Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.</a> J. Vis. Exp. (2016)
This video demonstrates the process for isolating spiral ganglion explants from the inner ear and co-culturing them with the organ of Corti on a multielectrode array. Spiral ganglia are carefully extracted and sectioned into smaller tissue sections or explants. These explants are cultured on the multielectrode array&#39;s active area to promote neuronal process outgrowth.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare Solutions for 
...

Neuronal Culture Techniques

Primary Neuronal Culture

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Source: Iyer, M. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a> J. Vis. Exp. (2023).
This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have ...

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

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