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.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+potassium+conductances+contribute+to+the+diverse+firing+properties+of+postnatal+mouse+vestibular+ganglion+neurons.">Heterogeneous potassium conductances contribute to the diverse firing properties of postnatal mouse vestibular ganglion neurons.</a> Journal of Neurophysiology. 96 (5), 2364-2376 (2006).</li><li>Iwasaki, S., Chihara, Y., Komuta, Y., Ito, K., Sahara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-voltage-activated+potassium+channels+underlie+the+regulation+of+intrinsic+firing+properties+of+rat+vestibular+ganglion+cells.">Low-voltage-activated potassium channels underlie the regulation of intrinsic firing properties of rat vestibular ganglion cells.</a> Journal of Neurophysiology. 100 (4), 2192-2204 (2008).</li><li>Cervantes, B., Vega, R., Lim&#243;n, A., Soto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity,+expression+and+functional+role+of+the+sodium-activated+potassium+current+in+vestibular+ganglion+afferent+neurons.">Identity, expression and functional role of the sodium-activated potassium current in vestibular ganglion afferent neurons.</a> Neuroscience. 240, 163-175 (2013).</li><li>Biel, M., Wahl-Schott, C., Michalakis, S., Zong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarization-activated+cation+channels:+From+genes+to+function.">Hyperpolarization-activated cation channels: From genes to function.</a> Physiological Reviews. 89 (3), 847-885 (2009).</li><li>Davis, R. 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A., Christov, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ionic Conductances of Vestibular Afferent Neurons: Shaping Head Motion Signals From the Inner Ear. , (2020).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+in+the+biophysical+properties+of+spiral-ganglion+and+vestibular-ganglion+neurons+in+neonatal+rats.">Similarities in the biophysical properties of spiral-ganglion and vestibular-ganglion neurons in neonatal rats.</a> Frontiers in Neuroscience. 15, 710275 (2021).</li><li>Armstrong, C. E., Roberts, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+frog+saccular+hair+cells:+distortion+by+enzymatic+dissociation.">Electrical properties of frog saccular hair cells: distortion by enzymatic dissociation.</a> The Journal of Neuroscience. 18 (8), 2962-2973 (1998).</li><li>Rocha-Sanchez, S. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+Kcnq4+in+vestibular+neurons+and+neurosensory+epithelia.">Developmental expression of Kcnq4 in vestibular neurons and neurosensory epithelia.</a> Brain Research. 1139, 117-125 (2007).</li><li>Meredith, F. L., Rennie, K. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+actions+of+brain-derived+neurotrophic+factor+and+neurotrophin-3+on+firing+features+and+ion+channel+composition+of+murine+spiral+ganglion+neurons.">Opposite actions of brain-derived neurotrophic factor and neurotrophin-3 on firing features and ion channel composition of murine spiral ganglion neurons.</a> The Journal of Neuroscience. 22 (4), 1385-1396 (2002).</li><li>Zhou, Z., Liu, Q., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+regulation+of+spiral+ganglion+neuron+firing+patterns+by+neurotrophin-3.">Complex regulation of spiral ganglion neuron firing patterns by neurotrophin-3.</a> The Journal of Neuroscience. 25 (33), 7558-7566 (2005).</li><li>Liu, X. -. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</td>
			<td>Zeiss&#160;</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&#160;</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&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L4386</td>
			<td>dissection and bath solutions&#160;</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&#160;</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 &#8211; 60 mg/kg dosing)&#160;</td>
			<td>MWI Animal Health</td>
			<td>15199</td>
			<td>for euthanasia</td>
		</tr>
		<tr>
			<td>PES membrane filters ,&#160; 0.2 micrometer&#160;</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&#160;</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&#160;</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&#160;</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&#62;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/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/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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

1.0K Views.  University of Southern California. 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.

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

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...