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 done so using acutely isolated hair cells removed from their native environment. In this article we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that expands upon these foundation studies.
While vestibular hair cells are relatively robust, the utility of the semi-intact preparation is critically dependent on the speed of excision. Typically, a preparation will remain viable at room temperature for a period of approximately 2-5 hr after excision. Therefore, minimizing the time taken from when the animal is euthanized to recording is paramount. The excision can be relatively easy and quick for a 3 week old mouse (5-7 min total), but gets progressively more difficult in an older animal (up to 25 min in 9 month old mice). In these older animals, dissection temperature is a critical determinant - by ensuring that the preparation is continually bathed in ice-cold oxygenated ACSF normal catabolic processes are inhibited.
As described in section 3.1 the preparation was continually perfused with oxygenated Leibovitz's medium, L-15, and whole-cell voltage clamp recordings were made using glass microelectrodes filled with KF internal solution 12. This combination of external and internal environments has been shown to provide more stable and longer- duration recordings than other potassium-based internals and/or Ringer's based extracellular solutions 13-14. This stability is crucial for the long protocols required to study electrophysiological properties and calcium dynamics of hair cells using patch-clamp and two-photon techniques respectively.
The fundamental objective of the semi-intact preparation is to provide a model for the study of vestibular hair cells that is disturbed as little as possible. While the preparation provides clear advantages over acutely isolated hair cells - for example, the full dynamics of whole cell currents are only revealed using the semi-intact preparation 3, 7- disruptions cannot be avoided. First, it is difficult to assess the impact of removing the "roof" of the ampulla (and presumably the attached cupula) on spontaneous hair cell signaling. Under normal physiological conditions, hair cells respond to bending of the cupula/hair bundle complex. Without this complex it is conceivable that spontaneous hair cell signaling may not be representative of in vivo conditions. Second, electrophysiological recordings in the semi-intact preparation are mostly limited to measurements of spontaneous activity or responses to simulated natural activity (i.e. direct current injections into the hair cell or mechanical bending of hair bundles). Therefore it is not possible to study the underlying hair cell properties in response to real-life activation (i.e. head movements). Nevertheless, the semi-intact preparation represents a new tool to help us understand hair cell function, and opens a number of avenues for future research.
One major advantage that the semi-intact preparation provides over acutely isolated hair cell preparations is that it allows simultaneous recordings from multiple hair cells and/or their associated primary afferents. Using dual, or multi-cell patch clamp recording techniques this feature provides a means of assessing directly hair cell/primary afferent signaling, something that cannot be achieved with traditional isolated preparations. In addition, using two-photon microscopy, the semi-intact preparation allows for simultaneous measurement of sub-cellular function across the full extent of the sensory epithelium. This means that information regarding calcium signaling and cellular metabolism can be accrued rapidly and in context. Finally, using mice allows for targeted genetic manipulation (e.g. green fluorescent protein tagged to calretinin expression as a marker for type I hair cells and calyx primary afferents). The semi-intact preparation provides a method to study the activity of hair cells and primary afferents differentially, and simultaneously. This alone, would provide a wealth of new information regarding how vestibular hair cells and primary afferents interact to detect and then encode information to be transmitted to the brain regarding head and body position.

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 border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</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></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </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 pronounced "sag" during depolarization and large "collapsing" tail currents in the type I hair cell, and the presence of transient inward sodium currents representing action potentials in the calyx primary afferent (Arrow in Figure 5C).
The preparation can also be used to quantify the impact of aging on individual vestibular hair cell types. Figure 6 compares the conductance (Figure 6A) and inactivation characteristics (Figure 6B) of type II vestibular hair cells in response to step depolarization from a negative resting potential in young and older mice. For the type II hair cells - the least sensitive subtype- significant differences were not observed in either parameter (see discussion).
In addition to the analysis of whole-cell properties, the semi-intact preparation also lends itself to large-scale analysis of the subcellular mechanisms underlying hair cell function. Figure 7 shows the differential localization of calcium to the type I hair cells and calyx primary afferents, as well as the sub cellular organization of calcium within these cells. Figure 7A shows the change in calcium fluorescence in an individual type I hair cell to the addition of 100 mM KCl. The time course of this change is plotted in Figure 7B. Importantly, the preparation allows for the investigation of hair cell activity at subcellular resolution, in multiple cells simultaneously, and in a "near normal" environment (Figure 7C)- a feature not possible using isolated hair cells and single cell electrophysiology.
<img src="/files/ftp_upload/50471/50471fig5.jpg" alt="Figure 5"/> 
Figure 5. Whole cell currents recorded from hair cells in the mouse vestibular sensory epithelium. A. Whole cell currents recorded from a type I vestibular hair cell characterized by large "collapsing" tail currents (Arrow). B. Whole cell currents recorded from a type II vestibular hair cell. Note the absence of tail currents. C. Whole cell recording from a calyx primary afferent characterized by transient sodium-mediated currents (Arrow) following depolarization from a negative resting potential (-120 mV). All cells were held at -60 mV.
<img src="/files/ftp_upload/50471/50471fig6.jpg" alt="Figure 6"/> 
Figure 6. Comparison of sensitivity and inactivation in type II hair cells from young and older mice. A. I/V relationships for type II hair cells recorded from young (1 month) and older (9 month) mice in response to increasing depolarization duration. B. The average conductance and inactivation ratio of type II hair cells in young and old mice based on the 100 msec I/V curves.
<img src="/files/ftp_upload/50471/50471fig7.jpg" alt="Figure 7"/> 
Figure 7. Two-photon calcium imaging of the semi-intact mouse vestibular sensory epithelium preparation. A. Top left panel) Two-photon micrographs showing BAPTA-1 Oregon Green calcium activation in response to normal L-15 perfusate (top) or L-15 containing 100 mM KCl (bottom). Arrows indicate an individual hair cell before and after the addition of KCl. B. Top right panel) Calcium fluorescence profile of the cell highlighted in A. C. Bottom panel) Two-photon micrograph of the sensory epithelium showing Oregon Green 488 BAPTA-1 loading of individual hair cells. Bottom panel Inset: subcellular localization of calcium to the type I hair cell cytoplasm (arrow) or the calyx primary afferent surrounding a type I hair cell (double arrows).

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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 ...

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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

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.