Funding for this work was provided by a Garnett Passe and Rodney Williams Memorial Foundation Project grant to R. Lim and A.J. Camp.

1. Animals
<ol>
	<li>Mice were obtained from the Australian Rodent Centre (ARC; Perth, Australia) and held at the University of Sydney Bosch Animal Facility on a normal 12-hr light/dark cycle with environmental enrichment. All experiments described were approved by The University of Sydney Animal Ethics Committee.</li>
	<li>Male and female mice (C57/Bl6) were used for all experiments since this strain are commonly used as the background for genetic manipulation, and can be considered equivalent to wildtype 8-9.</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Prepare 300 ml of a glycerol-based artificial cerebrospinal fluid (ACSF) consisting of (in mM): 26 NaHCO3, 11 glucose, 250 glycerol, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, and 2.5 CaCl2. Prior to the addition of CaCl2, gas the solution with carbogen (95% O2 and 5% CO2) to establish a pH of 7.4 and avoid calcium precipitation (cloudiness). Freeze the solution in a -80 &deg;C freezer for 45 min so that an ice slurry is formed.</li>
	<li>Deeply anaesthetize mice with ketamine (100 mg/kg) (Parnell, Alexandria, Australia) via an intra-peritoneal injection.</li>
	<li>Once hind-limb pinch reflexes are absent decapitate mice using sharp stainless steel scissors and make a sagittal skin incision using a razor blade (rounded #22) to expose the skull. At this point and throughout steps 2.3-2.8 the cranial vault, brain, and underlying vestibular apparatus should be kept as cool as possible by regular application of ice-cold ACSF over the tissue.</li>
	<li>Using the pointed arm of standard pattern scissors (FST, North Vancouver, Canada) make a small incision in the skull at Lambda and cut along the sagittal suture. Ensure that the brain is not "dragged" by the shear blade during this step.</li>
	<li>Carefully peel the parietal bones away laterally and the occipital bone posteriorly using shallow bend pearson rongeurs (FST).</li>
	<li>A small stainless steel spatula is then used to gently lift the brain from the surface of the middle and posterior cranial fossae, and the exposed vestibulocochlea nerve (CNVIII) cut midway between the inner ear and the brainstem with a pair of fine iris scissors. Cutting this nerve minimizes undue tension on axons of the primary afferents and their connections with hair cells.</li>
	<li>Following transection of CN VIII the brain is removed in toto.</li>
	<li>The vestibular labyrinth is now clearly visible in the middle cranial fossa, with the cochlea pointing anteromedially. Rongeur either side of the vestibular labyrinth before gently excising by gripping the anterior semicircular canal and pulling laterally.</li>
	<li>Immerse the excised labyrinths (Figure 1) in a dissecting dish containing the ice-cold, continuously gassed ACSF described in section 2.1. Under a stereomicroscope, hold the labyrinth to the base of the dish by gripping the cochlea. Use fine forceps to scratch away at the bone overlying the anterior semicircular canal ampulla. Once a small hole in the bone is achieved begin to flick the bone away from the ampulla. Caution should be taken not to push the forceps through the bone as this can cause damage to the underlying membranous labyrinth and sensory epithelium. Continue this procedure until both the anterior and adjacent horizontal semicircular membranous ducts and ampullae are exposed (Figure 2).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/50471/50471fig1.jpg" /> 
Figure 1. The isolated mouse vestibular labyrinth. A. Left panel) Schematic representation of the isolated mouse vestibular labyrinth. Important points of reference for accessing the vestibular sensory epithelium, the cochlea, anterior, and horizontal semicircular canals are labeled. Asterisks indicate the semicircular canal ampulla containing the vestibular sensory epithelium. B. Right panel) Photomicrograph of the isolated vestibular labyrinth from a 1-month-old mouse.
<img alt="Figure 2" src="/files/ftp_upload/50471/50471fig2.jpg" /> 
Figure 2. Exposure of the Membranous vestibular labyrinth. The bone overlying the anterior and horizontal semicircular canal ampullae have been scratched away to reveal the black/brown-speckled membranous ampullae and associated ampullary nerves (CNVIII). The schematic in the bottom panel represents the structures in the highlighted region of the photomicrograph and shows the relationship of the semicircular canal ducts to the ampullae and CNVIII.
<ol start="10">
	<li>Using fine forceps, gently lift the ampullae and associated utricle away from the bony labyrinth, ensuring that the central region of the ampullae (containing the sensory epithelium) is not damaged. In some cases the proximal part of the semicircular membranous duct may need to be cut with iris scissors to release the ampullae from the bone.</li>
	<li>Transfer the triad containing the two ampullae and utricle into a Petri dish filled with Leibovitz's L-15 culture medium (Sigma-Aldrich, St. Louis, MO). Use a fiber optic to "backlight" the tissue. This allows clear visualization of the crista containing the sensory epithelium within the ampullae.</li>
	<li>Carefully make an incision in the speckled "roof" of the utricle with the fine iris scissors. Continue this incision through the roof of the anterior and then the horizontal ampullae. Make the incision as close to the edge of the sensory epithelium as possible without contacting it (Figure 3A). Ensure that there are no pieces of membrane overlying the sensory epithelium (Figure 3B).</li>
	<li>Transfer the isolated semi intact preparation to a small glass-bottomed recording chamber filled with L-15 media. Weigh down the preparation using a grid of fine nylon fibers secured to a flattened U-shaped platinum wire (Figure 3B). Ideally, the fibers of the grid should not overlie the sensory epithelium, however in some cases where more stability is required, a single fiber transecting the sensory epithelium maybe preferred.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/50471/50471fig3.jpg" /> 
Figure 3. The isolated semi-intact preparation of the vestibular sensory epithelium. A. Schematic representation of the semi-intact preparation and electrode configuration. The ampulla overlying the crista has been "de-roofed" to expose the surface of the sensory epithelium (green). B. Photomicrograph of the semi-intact 'triad' preparation showing the anterior (ac) and horizontal (hc) crista (utricle obscured behind ac). Note the nylon fiber used to secure the preparation to the base of the recording chamber. Scale bar: 100 &mu;m. C. A recording electrode positioned on an individual vestibular hair cell. Scale bar: 15 &mu;m.
3. Electrophysiology
<ol>
	<li>Perfuse the semi-intact preparation with continuously oxygenated L-15 medium. The media contains a pH indicator (phenol red) and color of the medium should be monitored throughout the recordings. The pH with oxygen should be 7.3-7.4 and correspond to a red color.</li>
	<li>Prepare recording pipettes from 1.5 mm (1.19 mm ID) borosilicate glass using a two-step protocol (heat step 1: 72; and heat step 2: 50) on a micropipette puller (Narishige, Japan, model PP-830) to achieve a final impedance of 3-4 M&Omega;.</li>
	<li>Fill the shank of the pipette with 3-4 mm of Potassium fluoride-based internal solution containing (in mM) 110 KF, 12 KCl, 27 KOH, 1 NaCl, 10 HEPES, 10 EGTA, 1.8 MgCl2, 3 D-glucose, and 2 Na-ATP; pH 7.4 with KOH.</li>
	<li>Wrap the shank of the pipette 2-3 times and as far down to the tip as possible with a thin strip of parafilm to insulate the electrode and reduce pipette capacitance.</li>
	<li>Position the pipette over the sensory epithelium under low power magnification (5X) on an upright microscope (Olympus BX51). Switch to high power (40X) and visualize individual vestibular hair cells with an attached CCD camera.</li>
	<li>Position pipette on the membrane of a visualized hair cell (Figure 3C) using a micromanipulator (Sutter Instruments, California, USA). Once a gigaohm seal is achieved, rupture the cell membrane with a small amount of negative pressure applied through a suction port on the pipette holder.</li>
	<li>Make whole-cell voltage clamp recordings using standard techniques 8-9.</li>
</ol>
4. Two-photon Microscopy
<ol>
	<li>Prepare a 0.9% saline solution containing 5 mM Oregon Green 488 BAPTA-1 (OGB-1; hexapotassium salt; Invitrogen, Germany).</li>
	<li>While still submerged, place the "de-roofed" semi-intact triad preparation onto a small piece of filter paper (4X4 mm, 0.8 &mu;m thick; Millipore, Germany) ensuring that the sensory epithelium is unobstructed from above.</li>
	<li>Transfer the filter paper and preparation onto the saline covered base platinum electrode (7 &mu;l) of an electroporator consisting of the combination of a pulse generator and a wide-band amplifier, and cover with a further 5 &mu;l of 5 mM solution of the synthetic calcium indicator dye Oregon Green 488 BAPTA-1 (Figure 4).</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/50471/50471fig4.jpg" /> 
Figure 4. Bulk electroporation. A. schematic showing cross-section of electroporation configuration. The semi-intact preparation (*) is placed on a piece of filter paper immersed in calcium dye (Oregon Green 488 BAPTA-1) and current applied between 2 platinum electrodes. Adapted from Briggman and Euler, 2011 10.
<ol start="4">
	<li>Bring the top platinum electrode parallel with the base electrode at a distance of approximately 2 mm, being careful not to disrupt the orientation of the preparation when contact with the Oregon Green 488 BAPTA-1 solution is made.</li>
	<li>Pass a brief current pulse across the preparation (Parameters for the current; +13 V, 10 msec pulse width, 1 Hz frequency, 10 square-wave pulses) 10-11.</li>
	<li>On the bottom of a glass bottomed recording chamber filled with L-15 medium, place a small dab of silicon oil and position the semi-intact preparation onto it, again ensuring that the sensory epithelium is unobstructed from above. To ensure stability throughout imaging, replace the nylon grid over the preparation as described above (see point 2.13).</li>
	<li>Using the two-photon microscope make optical recordings of spontaneous calcium activity in the sensory epithelium (Figure 7) using standard imaging protocols 10-11.</li>
</ol>

<ol>
	<li>Dulon, D., Safieddine, S., Jones, S. M., Petit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otoferlin+is+critical+for+a+highly+sensitive+and+linear+calcium-dependent+exocytosis+at+vestibular+hair+cell+ribbon+synapses.">Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses.</a> J. Neurosci. 29, 10474-10487 (2009).</li><li>Highstein, S., Art, J., Holstein, G., Rabbitt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+pre-+and+post-synaptic+recording+from+the+peripheral+vestibular+calyx+and+its+included+type+I+hair+cell.">Simultaneous pre- and post-synaptic recording from the peripheral vestibular calyx and its included type I hair cell.</a> , (2009).</li><li>Kindig, A. E., Lim, R., Callister, R. J., Brichta, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+dependent+currents+in+type+I+and+II+hair+cell+and+calyx+terminals+of+primary+afferents+in+an+intact+in+vitro+mouse+vestibular+crista+preparation.">Voltage dependent currents in type I and II hair cell and calyx terminals of primary afferents in an intact in vitro mouse vestibular crista preparation.</a> , (2009).</li><li>Chatlani, S., Goldberg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+recordings+from+calyx+endings+in+the+turtle+posterior+crista.">Whole-cell recordings from calyx endings in the turtle posterior crista.</a> , (2010).</li><li>Songer, J. E., Eatock, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+in+the+mammalian+saccule.">Transduction in the mammalian saccule.</a> , (2010).</li><li>Lee, H. Y., Camp, A. J., Callister, R. J., Brichta, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+primary+afferent+activity+in+an+in+vitro+preparation+of+the+mouse+inner+ear.">Vestibular primary afferent activity in an in vitro preparation of the mouse inner ear.</a> J. Neurosci. Methods. 145, 73-87 (2005).</li><li>Lim, R., Kindig, A. E., Donne, S. W., Callister, R. J., Brichta, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+accumulation+between+type+I+hair+cells+and+calyx+terminals+in+mouse+crista.">Potassium accumulation between type I hair cells and calyx terminals in mouse crista.</a> Exp. Brain Res. 210, 607-621 (2011).</li><li>Camp, A. J., Callister, R. J., Brichta, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+synaptic+transmission+differs+in+mouse+type+A+and+B+medial+vestibular+nucleus+neurons+in+vitro.">Inhibitory synaptic transmission differs in mouse type A and B medial vestibular nucleus neurons in vitro.</a> J. Neurophysiol. 95, 3208-3218 (2006).</li><li>Camp, A. J., 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=Attenuated+glycine+receptor+function+reduces+excitability+of+mouse+medial+vestibular+nucleus+neurons.">Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons.</a> Neuroscience. 170, 348-360 (2010).</li><li>Briggman, K. L., Euler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+electroporation+and+population+calcium+imaging+in+the+adult+mammalian+retina.">Bulk electroporation and population calcium imaging in the adult mammalian retina.</a> J. Neurophysiol. 105, 2601-2609 (2011).</li><li>Briggman, K. L., Helmstaedter, M., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiring+specificity+in+the+direction-selectivity+circuit+of+the+retina.">Wiring specificity in the direction-selectivity circuit of the retina.</a> Nature. 471, 183-188 (2011).</li><li>Rennie, K. J., Streeter, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+currents+in+isolated+vestibular+afferent+calyx+terminals.">Voltage-dependent currents in isolated vestibular afferent calyx terminals.</a> J. Neurophysiol. 95, 26-32 (2006).</li><li>Hudspeth, A. J., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+voltage-+and+ion-dependent+conductances+in+saccular+hair+cells+of+the+bull-frog,+Rana+catesbeiana.">Kinetic analysis of voltage- and ion-dependent conductances in saccular hair cells of the bull-frog, Rana catesbeiana.</a> J. Physiol. 400, 237-274 (1988).</li><li>Rennie, K. J., Ashmore, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+currents+in+isolated+vestibular+hair+cells+from+the+guinea-pig+crista+ampullaris.">Ionic currents in isolated vestibular hair cells from the guinea-pig crista ampullaris.</a> Hear. Res. 51, 279-291 (1991).</li></ol>

The authors declare that they have no competing financial interests.

The mechanisms underlying our sense of balance have received limited attention in comparison with other sensory systems, e.g. the visual and auditory systems. Of the studies that have investigated changes in vestibular or balance function, most have focused on behavioral measures including the vestibulo-ocular reflex, with incomplete knowledge of the fundamental building blocks of balance- the vestibular hair cells themselves. Those studies that have concentrated on the hair cells have almost invariably do...

Despite the significant contribution of the vestibular system to our everyday lives, a clear understanding of the processes responsible for the observed decline in vestibular function with age remain elusive. One reason for this lack of knowledge is that decline in vestibular function has almost exclusively been explored using behavioral assays, including the vestibulo-ocular reflex (VOR), a precise indicator of extrinsic vestibular function, but provides limited insights into the changes of intrinsic components. This is a major impediment to our understanding of vestibular hair cell function in health, disease, or aging.
While there have been many studies of individual vestibular hair cells, a major shortcoming has been the reliance on acute hair cell preparations, where hair cells and even calyx afferent terminals are removed from their normal environment via mechanical and/or enzymatic treatment. Such approaches inevitably disrupt the delicate microarchitecture between hair cell and calyx, and hair cell and supporting cell. With the development of semi-intact preparations 1-5, and an isolated mouse labyrinth preparation 6, there is now an opportunity to study the various forms of synaptic communication under conditions that more closely resemble those in vivo. Indeed, Lim et al. (2011) showed marked differences in whole cell currents recorded from acutely isolated type I vestibular hair cells compared to those that remained embedded within the neuroepithelium. Specifically, potassium is thought to accumulate in the intercellular space, between the hair cell and calyx afferent, and significantly alter hair cell response7. This type of information would be impossible to obtain without the semi-intact preparation of the vestibular sensory epithelium described here. We demonstrate the semi-intact preparation of the mouse crista 3, and show representative results obtained from whole-cell patch electrophysiology, and two-photon calcium imaging.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Leibovitz medium L-15</td><td>Sigma Aldrich</td><td>L4386-10X1L</td><td></td></tr><tr><td>BAPTA-1-oregon green</td><td>Invitrogen</td><td>O6806</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT &lt;/strong&gt;</td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>A60S</td><td></td></tr><tr><td>Upright microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>Two-photon microscope</td><td>Olympus/La Vision</td><td>BX51WI/ TriMScope II</td><td></td></tr><tr><td>Dumont #5 SF Forceps</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>Friedman-Pearson Rongeurs</td><td>FST</td><td>16221-14</td><td></td></tr><tr><td>Standard Pattern Scissors</td><td>FST</td><td>14001-12</td><td></td></tr><tr><td>InstraTECH A-D converter</td><td>HEKA</td><td>ITC-18</td><td></td></tr><tr><td>Sutter Micromanipulator</td><td>Sutter</td><td>MP-225/M</td><td></td></tr><tr><td>multiclamp amplifier</td><td>Axon Instruments</td><td>700B</td><td></td></tr><tr><td>Data acquisition software (electrophysiology)</td><td>Axograph</td><td>N/A</td><td></td></tr><tr><td>Imspector Data acquisition software (two-photon)</td><td>Max Planck innovation</td><td>N/A</td><td></td></tr></tbody></table>

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.

The electrophysiological properties of vestibular hair cells are dependent on the complex microarchitecture within which they are embedded 7. Figure 5 shows that the semi-intact vestibular epithelium preparation can be used to differentiate between type I hair cells (Figure 5A), type II hair cells (Figure 5B), and the calyx primary afferent (Figure 5C) based on characteristic whole cell conductances. These characteristics include a prono...

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

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 ...

an-isolated-semi-intact-preparation-mouse-vestibular-sensory

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.8K Views.  University of Sydney. 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.

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

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biology

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioengineering

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

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 ...

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 ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. ...

Medicine

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

Source: Tu, H., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/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

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JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear

Source: Iyer, M. R. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a>&#160;J. Vis. Exp. (2023).
This video showcases a method for isolating vestibular neurons from a mouse pup&#39;s inner ear. It meticulously outlines the steps involved in dissecting the inner ear, harvesting the vestibular ganglion, and subsequently dissociating the ganglion into single cells. These cells are then cultured on glass-bottom dishes to establish a vestibular neuron culture.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparations
NOTE: The solutions ...

Neuronal Culture Techniques

Primary Neuronal Culture

Preparation of Mouse Vomeronasal Organ for Vibratome Sectioning

Source: Ackels, T., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54517">In-depth physiological analysis of defined cell populations in acute tissue slices of the mouse vomeronasal organ.</a> J. Vis. Exp. (2016)
This video outlines the isolation of a mouse vomeronasal organ (VNO) and tissue preparation for vibratome sectioning. Sections are used to characterize the biophysical and physiological attributes of a specific group of sensory neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
Both ...

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

Summary

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Chapters in this video

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Optical Imaging of Isolated Murine Ventricular Myocytes

Cochlear Surface Preparation in the Adult Mouse

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

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

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

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

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear

Preparation of Mouse Vomeronasal Organ for Vibratome Sectioning