The authors wish to thank Dr. Candace Hsieh for suggestions and assistance with imaging techniques and Dr. Doris Wu for expertise on chick inner ear anatomy during early embryogenesis. This work was supported by NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796, and DOE GAANN P200A120165.

1. Prepare the Following Dissection Tools and Reagents
<ul>
	<li>Artificial cerebrospinal fluid (aCSF; 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 20 mM NaHCO3, 3 mM HEPES, 10 mM Glucose, 2 mM CaCl2, 1.3 mM MgSO4) continuously infused with 95% O2 / 5% CO2 at room temperature. For infusion, fill to 2/3 a 500 ml wide-mouth Nalgene jar with a hole drilled in the lid. Tank will be attached by tubing to a glass stem bubbler, which penetrates the aCSF through the hole in the jar lid. Adjust pressure to obtain a constant stream of bubbles that reach about 1/3 of the liquid but are not forceful enough to cause splashing over the rim. Submerge 5 ml glass vials (up to 6) into the Nalgene jar to separate individual samples from each other and from the gas flow turbulence. ACSF should be oxygenated for at least 20 min prior to any tissue incubation.</li>
	<li>Small silicone dissection dish (35 mm x 10 mm Petri coated using Sylgard Elastomere Kit) with two dissection pins placed into small hexagonal polystyrene (47 mm bottom) weigh boat.</li>
	<li>Two fine-tip forceps, one curved-tip forceps, a 50 ml beaker, and a container for tissue waste.</li>
	<li>Pulled glass micropipette (1.2 OD / 0.9 ID) broken to approximately 20-50 &mu;m opening at the tip and filled with rhodamine dextran amine (RDA, MW = 3,000, Invitrogen) in a 6.25% solution with 0.4% Triton X-100 in PBS. Attach by fine tubing to picospritzer and stabilize to micromanipulator.</li>
</ul>
2. Micro-dissection to Reveal Basilar Papilla at E8 4,5
<ol>
	<li>With curved forceps, cut E8 embryo just below the brainstem, placing head directly onto silicone-coated dissection dish within weigh boat.</li>
	<li>Using dissection pins, position the head with a ventral view so the ear on the side of interest is slightly visible, the head tilted slightly dorsally and pointing 10-30 degrees away from center (Figure 2A). Immediately secure the head with pins. If bilateral tracing is desired, center the head at 0 degrees, or a full ventral view so both sides can be accessed. Similarly, for a left-sided injection, secure the head at -10 to -30 degrees from center so the left side is accessible.</li>
	<li>Using the 50 ml beaker, transfer 30 ml of oxygenated aCSF into the dish, fully submerging the tissue.</li>
	<li>Gently grasp and peel down the lower jaw using fine-tip forceps, then remove the lower eyelid on the side of interest.</li>
	<li>Remove the overlying vascular tissue, including any remaining palate and gullet. These soft tissues should peel away revealing the smooth surface of developing chondrocranium with the distinctive vertebral arteries at its surface. By this point, the characteristic white otoconial mass should be visible.</li>
	<li>Carefully dissect the skin around the external meatus, cartilage of the jaw joint, and the middle ear while leaving the inner ear intact. Use the forceps to gently cut away these cartilaginous structures.</li>
	<li>Remove vertebral arteries and pooling blood using a gentle sweeping motion with the fine forceps tips. Blood can obscure view and coagulation may plug the injection pipette opening.</li>
	<li>The cochlear duct with adjacent sensory epithelium (basilar papilla) and acoustic ganglion lies directly beneath the chondrocranium 6 and can be visualized as an elongated finger-like projection extending from the inner ear ventrally, with the distal-most tip (apex) near the anterior midline 5. There may be a small pooling of blood (red) from the dissection and/or otolithic calcifications (white) at the apex of the cochlear duct (Figure 2B) that can aid in visualization. At the apex is the lagenar macula, a sensory patch thought to be of vestibular function 7,8.</li>
</ol>
3. Selective Labeling of CN VIII Auditory Fibers
<ol>
	<li>Place the injection pipette near the embryo surface at the cochlear duct and basilar papilla region.</li>
	<li>Slowly lower the micropipette to first puncture the cranium. This cranial region is thinner than surrounding areas and requires less force to penetrate. The micropipette should now be inside the cochlear duct. Optional: a small injection of tracing dye can be made here to aid in visualizing the basilar papilla.</li>
	<li>Without repositioning the micropipette, continue lowering until it just breaches the deep border of the cochlear duct (Figure 1). Perform an injection here and repeat in multiple regions along the length of the basilar papilla (Figures 2C, 2D), excluding the most proximal tip where the vestibular lagena is located. With the picospritzer set between 10-30 psi, several injections are made. The volume of injected dye fluid varies, and depends on the extent of labeling desired. Each injection should result in a fluorescent dye-labeled spot of approximately 200 - 400 &mu;m diameter (Figure 2D).</li>
	<li>Once the injection is complete, use forceps to transect the sample above the brainstem and use a transfer pipette to place the caudal portion into a small jar within the container of aCSF continually being perfused with 95% O2 / 5% CO2.</li>
	<li>For each embryo, allow 20-30 min for dye transfer, depending on the distance of central tracing desired.</li>
	<li>Remove tissue and prepare for histological sectioning. In the example shown here, tissue is immersed in 4% paraformaldehyde (in 1X PBS) overnight, 30% sucrose (in 1X PBS) overnight. </li>
</ol>
4. Counterstaining and Analysis
<ol>
	<li>Counterstaining is useful to help orient the observer to anatomical location as well as identify particular regions of interest. An effective counterstain in this preparation is neurofilament immunolabeling (Figures 1, 3B, 3E), which strongly labels axons and allows for demarcation of axonal tracts in the CNS.</li>
	<li>Analysis should be performed throughout the labeled region, as the projections can extend several hundred microns or more along the rostrocaudal axis. Some examples of basic analyses are: targeting error rates, timing and/or changes in divergence of projection patterns. RDA-labeled axons are visualized in the peripheral and central nervous system with high resolution (Figures 3A, 3D).</li>
</ol>

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No conflicts of interest are declared.

Studies of the early development of the VIIIth nerve have been limited in part because of the difficulty in identifying embryonic axons arising from multiple distinct ganglia. Several studies have explored the molecular signals guiding auditory and vestibular sensory cell and ganglion cell fates during early development, 5,11,12 but the processes regulating central innervation have yet to be determined. Reports of acoustic ganglion cell projections typically describe peripheral processes to sensory epit...

<table><tbody><tr><td>Polystyrene Weigh Dish</td><td>Fisher Scientific</td><td>02-202-101</td><td></td></tr><tr><td>Petri Dish, 35 X 10 mm</td><td>Fisher Scientific</td><td>50820644</td><td>Use to make silicone dissection dish</td></tr><tr><td>Sylgard Silicone Elastomer Kit</td><td>World Precision Instruments</td><td>SYLG184</td><td>Coat Petri to make dissection dish</td></tr><tr><td>Dissection Pins</td><td>Various</td><td></td><td>Holds embryo in place during dissection</td></tr><tr><td>NaCL</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>KCl</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>Glucose</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Various</td><td></td><td>part of aCSF recipe</td></tr><tr><td>Container for aCSF. Suggest translucent wide-mouth Nalgene jar, 500 ml (16 oz) with lid.</td><td>CPLabSafety</td><td>QP-PLC-03717</td><td>Drill hole opening in top of lid for glass bubling stem to penetrate liquid</td></tr><tr><td>Empty 5 ml glass vial or comparable transparent vial</td><td>American Pharmaceutical Partners, Inc</td><td>6332300105</td><td>Use during aCSF incubation to keep samples separate from each other and from the bubbling stream</td></tr><tr><td>Tank of carbogen (95%O&lt;sub&gt;2&lt;/sub&gt; / 5%CO&lt;sub&gt;2&lt;/sub&gt;) connected by tubing to bubbler</td><td>Various</td><td></td><td>Attach by tubing to glass stem bubbler for infusion into aCSF</td></tr><tr><td>Glass stem bubbler</td><td>Various</td><td></td><td>To infuse carbogen into aCSF</td></tr><tr><td>Curved-tip forceps</td><td>World Precision Instruments</td><td>501008</td><td>To remove embryo head from egg</td></tr><tr><td>Two fine-tip forceps</td><td>World Precision Instruments</td><td>501985</td><td>For micro-dissection</td></tr><tr><td>50 ml Beaker</td><td>various</td><td></td><td></td></tr><tr><td>Rhodamine Dextran Amine (RDA)</td><td>Invitrogen</td><td>various</td><td>Fluorescent axon tracer</td></tr><tr><td>Triton X-100</td><td>ICN Biomedicals</td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline, (1X PBS)</td><td>Various</td><td></td><td>Standard lab reagent</td></tr><tr><td>Thin Wall Glass Capillaries, 1.2 OD, .9 ID 4&quot; (100 mm) length</td><td>World Precision Instruments</td><td>TW120F-4</td><td>Load with RDA. Each capillary makes two glass micropipettes</td></tr><tr><td>Needle / Pipette puller</td><td>David Kopf Instruments</td><td>Model 720</td><td>Settings used: Heat 16.4, Solenoid 2.2</td></tr><tr><td>Picospritzer</td><td>Parker Instrumentation</td><td>various</td><td>Attach by fine tubing to glass micropipette</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>various</td><td></td></tr><tr><td>Dissection microscope with fluorescence</td><td>Various</td><td></td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Various</td><td></td><td>Standard lab reagent</td></tr><tr><td>anti-Neurofilament antibody, optional</td><td>Millipore</td><td>AB1991</td><td>Follow histological protocol recommended by manufacturer</td></tr><tr><td>Cryostat and associated materials for sectioning</td><td>Leica</td><td>various</td><td></td></tr><tr><td>Epifluorescent microscope for imaging</td><td>Zeiss, various</td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

The components of the VIIIth nerve and the anatomy of the nerve itself are complex and convoluted (Figures 1, 3). By selectively tracing fibers arising from acoustic ganglion cells, segments of the VIIIth nerve as well as primary auditory afferents within the brainstem can be cleanly traced and distinguished from their vestibular counterparts (Figures 2, 3). Likewise, this technique could be used to study peripheral projections of the acoustic ganglion cells (Figure 3G),...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K Views.  University of California, Irvine. Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Video: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Developmental Biology

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

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

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

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

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 ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

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Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

Preparation and Culture of Chicken Auditory Brainstem Slices

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

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