עבודה זו נתמכה על ידי מחקר לחירשות קרן מחקר מענק EY ו NIDCD DC006476 אל EG ועל ידי NIDCD DC005211 למרכז השמיעה ושיווי המשקל, מאוניברסיטת ג&#39;ונס הופקינס. אמנות זכויות יוצרים טים פלפס, מאוניברסיטת ג&#39;ונס הופקינס. LG כתב את כתב היד הראשוני; EY ו-LG צילם את לנתיחה ואת ההקלטה. כל המחברים בתנאי דמויות מופת ותרמו בכתב היד.

<ol>
	<li>Glowatzki, E., Fuchs, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitter+release+at+the+hair+cell+ribbon+synapse.">Transmitter release at the hair cell ribbon synapse.</a> Nat Neurosci. 5 (2), 147-154 (2002).</li><li>Goutman, J. D., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+calcium+dependence+of+transmitter+release+at+a+single+ribbon+synapse.">Time course and calcium dependence of transmitter release at a single ribbon synapse.</a> Proc Natl Acad Sci U S A. 104 (41), 16341-16346 (2007).</li><li>Yi, E., Roux, I., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+HCN+channels+shape+excitatory+postsynaptic+potentials+at+the+inner+hair+cell+afferent+synapse+in+the+mammalian+cochlea.">Dendritic HCN channels shape excitatory postsynaptic potentials at the inner hair cell afferent synapse in the mammalian cochlea.</a> J Neurophysiol. 103 (5), 2532-2543 (2010).</li><li>Grant, L., Yi, E., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+release+shape+the+postsynaptic+response+at+the+inner+hair+cell+ribbon+synapse.">Two modes of release shape the postsynaptic response at the inner hair cell ribbon synapse.</a> J Neurosci. 30 (12), 4210-4220 (2010).</li><li>LoGiudice, L., Matthews, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ribbons+at+sensory+synapses.">The role of ribbons at sensory synapses.</a> Neuroscientist. 15 (4), 380-391 (2009).</li><li>Singer, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+multivesicular+release+at+a+mammalian+ribbon+synapse.">Coordinated multivesicular release at a mammalian ribbon synapse.</a> Nat Neurosci. 7 (8), 826-833 (2004).</li><li>Keen, E. C., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+characteristics+of+the+hair+cell's+afferent+synapse.">Transfer characteristics of the hair cell's afferent synapse.</a> Proc Natl Acad Sci U S A. 103 (14), 5537-5542 (2006).</li><li>Li, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unitary+event+underlying+multiquantal+EPSCs+at+a+hair+cell's+ribbon+synapse.">The unitary event underlying multiquantal EPSCs at a hair cell's ribbon synapse.</a> J Neurosci. 29 (23), 7558-7568 (2009).</li><li>Singer, J. H., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+depletion+and+synaptic+depression+at+a+mammalian+ribbon+synapse.">Vesicle depletion and synaptic depression at a mammalian ribbon synapse.</a> J Neurophysiol. 95 (5), 3191-3198 (2006).</li><li>Suryanarayanan, A., Slaughter, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+mediated+by+internal+calcium+stores+in+rod+photoreceptors.">Synaptic transmission mediated by internal calcium stores in rod photoreceptors.</a> J Neurosci. 26 (6), 1759-1766 (2006).</li><li>Neef, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+mechanism+of+exocytosis+at+the+hair+cell+ribbon+synapse.">Probing the mechanism of exocytosis at the hair cell ribbon synapse.</a> J Neurosci. 27 (47), 12933-12944 (2007).</li><li>Tritsch, N. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+spontaneous+activity+in+the+developing+auditory+system.">The origin of spontaneous activity in the developing auditory system.</a> Nature. 450 (7166), 50-55 (2007).</li><li>Kros, C. J., Ruppersberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+potassium+current+in+inner+hair+cells+during+development+of+hearing+in+mice.">Expression of a potassium current in inner hair cells during development of hearing in mice.</a> Nature. 394 (6690), 281-284 (1998).</li><li>Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+of+frequency+representation+in+the+rat+cochlea.">Developmental changes of frequency representation in the rat cochlea.</a> Hear Res. 56 (1-2), 1-7 (1991).</li><li>Seal, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorineural+deafness+and+seizures+in+mice+lacking+vesicular+glutamate+transporter+3.">Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3.</a> Neuron. 57 (2), 263-275 (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

צעד קריטי בהליך זה הוא לנתיחה. אם הרקמה נמתחת או ניזוקו במהלך דיסקציה, סיבים מביא לא ישרדו. רקמות של חולדות צעירות הוא אלסטי יותר סלחני. אנו מוצאים כי ימים לאחר הלידה 10-11 הם הקלה ביותר לנתח וניסויים יש שיעור הצלחה גבוה יותר. תואר משמעותי של התבגרות שבלול מתרחשת לאחר הלידה, עם חולדות מתחילים לשמוע סביב יום הלידה 12 14. לכן, בגיל שבו דיסקציה היא הקלה ביותר, סינפסות לא יכול להיות בוגר מלא 4. דיסקציה המתואר כאן לחולדות הוא בעיקרו זהה עבור עכברים, ההבדל העיקרי הוא בגודל קטן יותר של שבלול העכבר. טכניקה זו מאפשרת את התכונות של סינפסות סרט להיבדק אצל עכברים שונה transgenically 15. שינויים בהמשך טכניקה זו כוללים: הוספת צבע פלואורסצנטי לפתרון תאיים לסיבי תווית 3; הקלטות יחד עם תא שיער presynaptic הפנימית באוטון מביא postsynaptic, המאפשר להעביר פונקציה בין תאים הסינפטי לפני ואחרי שייקבע 2 ומשוחרר- חותם הקלטות תאיים על boutons מביא כדי למנוע אובדן של שלמות התא. תצורת הקלטה תאיים קל יותר להגיע מאשר בתצורת כולו התא ניסויים בדרך כלל לאורך זמן רב יותר.

לעיל הוא ציוד אלקטרו setups שלנו המשמש הקלטה מ מסופי מביא.

postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse

סינפסה בין תא מביא את השיער הפנימי (IHC) לבין סיבי עצב השמיעה מספק האתר נגיש electrophysiologically הקלטה הפעילות postsynaptic של סרט בודד סינפסה 1-4. סינפסות רצועת הכלים של תאים חושית שחרור הנוירוטרנסמיטר ברציפות, קצב אשר מווסתת בתגובה לשינויים מדורגים בפוטנציאל הממברנה IHC 5. סינפסות רצועת הוכחו פועלים על ידי שחרור multivesicular, שם שלפוחית ​​מרובות ניתן לשחרר זמנית לעורר זרמים postsynaptic מעוררים (EPSCs) משתנה אמפליטודות 1, 4, 6-11. גם התפקיד של הסרט presynaptic, ולא את המנגנון הבסיסי לשחרר multivesicular מובנת כיום היטב. IHC היא מעוצבבים על ידי סיבי עצב השמיעה 10-20, וכל אנשי הקשר הסיב IHC עם אחד unmyelinated סיום להקים סינפסה סרט בודד. גודלו הקטן של IHCs boutons מביא קשר (כ 1 מיקרומטר קוטר) מאפשר הקלטות עם רזולוציה טמפורלית חריגים להתבצע. יתר על כן, הטכניקה ניתן להתאים שיא משני תאים טרום postsynaptic בו זמנית, המאפשר להעביר פונקציה ב הסינפסה להיחקר ישירות 2. שיטה זו ולכן מספק שבאמצעותו היבטים בסיסיים של עצבית ניתן ללמוד, לשחרר multivesicular לתפקד החמקמק של הסרט בתאי חישה.

Watch this Scientific Journal Video about Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse at JoVE.com

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Whole-cell-clamp תיקון הקלטות של עצב השמיעה דנדריטים סיבים בבית סינפסה סרט פנימי תא שיער שבלול יונקים.

הקלטות Postsynaptic ב דנדריטים מביא פנייה שבלול תאים שיער פנימית: שחרור Multivesicular ניטור ב סינפסה סרט

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

postsynaptic-recordings-at-afferent-dendrites-contacting-cochlear

Research

JoVE Journal

Neuroscience

18.4K Views.  The Johns Hopkins School of Medicine. Whole-cell-clamp תיקון הקלטות של עצב השמיעה דנדריטים סיבים בבית סינפסה סרט פנימי תא שיער שבלול יונקים.

Video: Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp מדידות קיבול ו Ca 2 + הדמיה על מסופי עצב יחיד ב Slices רשתית

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

הקלטה משולב של תוויות מסוף פלט ועצב מבחינה מכנית מאולצת מביא ב Endings עכבר זקיק השיער אזמלי

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

תיוג דקטרן וספיגת בתאי שיער מורורין בשידור חי ופונקציונלי

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

Bioengineering

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

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

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

גזע המוח השמיעתית תגובה והקלטה החיצוני תא שיער תיקון שלם-תא קלאמפ בחולדות כמחנכת

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

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

מורפולוגית ופונקציונלית הערכה של הסרט סינפסות בתדר ספציפי אזורים של העכבר שבלול

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

הדמיית חבילת סטריאוסיליה ברזולוציה ננומטרית בתאי שיער שמיעתיים של יונקים חיים

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The most common method for detecting the loss of ribbon synapses is immunolabeling because it allows for quantitative sampling from several tonotopic locations in an individual cochlea. However, the structures of interest are buried deep inside the bony cochlea. Gerbils are used as an animal model for age-related hearing loss. Here, routine protocols for fixation, immunolabeling gerbil cochlear whole mounts, confocal imaging, and quantifying ribbon synapse numbers and volumes are described. Furthermore, the particular challenges associated with obtaining good material from valuable aging individuals are highlighted. 
Gerbils are euthanized and either perfused cardiovascularly, or their tympanic bullae are carefully dissected out of the skull. The cochleae are opened at the apex and base and directly transferred to the fixative. Irrespective of the initial method, the cochleae are postfixed and subsequently decalcified. The tissue is then labeled with primary antibodies against pre- and postsynaptic structures and hair cells. Next, the cochleae are incubated with secondary fluorescence-tagged antibodies that are specific against their respective primary ones. The cochleae of aged gerbils are then treated with an autofluorescence quencher to reduce the typically substantial background fluorescence of older animals' tissues. 
Finally, cochleae are dissected into 6-11 segments. The entire cochlear length is reconstructed such that specific cochlear locations can be reliably determined between individuals. Confocal image stacks, acquired sequentially, help visualize hair cells and synapses at the chosen locations. The confocal stacks are deconvolved, and the synapses are either counted manually using ImageJ, or more extensive quantification of synaptic structures is carried out with image analysis procedures custom-written in Matlab.

A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

סינפסות סרט אימונו-לבלים וספירתם בשבלול גרביל הצעיר והזקן

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The ...

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.

בידוד וטיפוח סומאטה גנגליון ווסטיבולרי וספירלי ממכרסמים יילודים להקלטות טלאי

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

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicles

Source: Bewick, G. et al., <a href="https://app.jove.com/v/53854">Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings.</a> J. Vis. Exp. (2016).
This video demonstrates the combined recording of mechanically stimulated afferent output and nerve terminal labeling in the hair follicles of a mouse pinna. It outlines the steps involved in the mechanical stimulation of hair follicle nerve terminals before and after exposure to a fluorescent membrane dye, electrophysiology recording of the generated action potentials, and visualization of the nerve terminal labeling in the hair follicles.

הקלטה משולבת של תפוקת Afferent מגורה מכנית ותיוג מסוף עצבי בזקיקי שיער עכבר

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

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Hair Cells

Hair cells are the sensory receptors of the auditory system&mdash;they transduce mechanical sound waves into electrical energy that the nervous system can understand. Hair cells are located in the organ of Corti within the cochlea of the inner ear, between the basilar and tectorial membranes. The actual sensory receptors are called inner hair cells. The outer hair cells serve other functions, such as sound amplification in the cochlea, and are not discussed in detail here.

Hair cells are named after the hair-like stereocilia that protrude from their tops and touch the tectorial membrane. The stereocilia are arranged by height and are attached by thin filaments called tip links. The tip links are connected to stretch-activated cation channels on the tips of the stereocilia.

When a sound wave vibrates the basilar membrane, it creates a shearing force between the basilar and tectorial membranes that moves the hair cell stereocilia from side to side. When the cilia are displaced towards the tallest cilium, the tip links stretch, opening the cation channels. Potassium (K+) then flows into the cell, because there is a very high concentration of K+ in the fluid outside of the stereocilia. This large voltage difference creates an electrochemical gradient that causes an influx of K+ once the channels are opened.

This influx of positive charge depolarizes the cell, increasing the voltage across the membrane. This causes voltage-gated calcium (Ca2+) channels in the cell body to open, and Ca2+ flows into the cell. Ca2+ triggers a signaling cascade that causes synaptic vesicles containing excitatory neurotransmitter molecules to fuse to the cell membrane and be released, exciting the postsynaptic auditory nerve cell and increasing the transmission of action potentials to the brain. When the stereocilia are pushed in the opposite direction, towards the shortest stereocilia, the tip links relax, the cation channels close, and the cell becomes hyperpolarized (i.e., the membrane potential is more negative) compared to its resting state.

Characteristics of the sound wave, such as frequency, are encoded in the pattern of hair cell activation and, consequently, auditory nerve cell activation. This information is then sent to the brain for interpretation.

Hair Cells in Auditory System

תאי שיער

Hair cells are the sensory receptors of the auditory system&mdash;they transduce mechanical sound waves into electrical energy that the nervous system can ...