We would like to thank Drs. Leslayann Schecterson, Yuan Wang, Andres Barria and Mrs. Ximena Optiz-Araya for initial assistance with protocol set-up and for providing plasmids. This work was supported by NIH/NIDCD grant DC013841 (JTS).

All procedures were approved by Northwestern University Institutional Animal Care and Use Committees, and carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
1. Egg Handling
<ol>
	<li>Purchase fertilized eggs from a local vendor. Store eggs at 13 &#176;C in a refrigerator for no longer than 5 days prior to incubation. Embryo viability significantly decreases after 1 week.</li>
	<li>Swab each egg with 70% ethanol before setting in the incubator.</li>
	<li>Set 1-2 dozen(s) of eggs on their sides in an incubator with the temperature set to 38 &#176;C at ~50% humidity. This ensures proper embryo positioning for electroporation and optimal viability, respectively. 
	NOTE: After 48-52 h of incubation, the eggs will be at Hamburger-Hamilton (HH) stage ~12-13 and ready for electroporation.</li>
</ol>
2. Preparation
<ol>
	<li>Plasmid Mixing
	<ol>
		<li>Add 1 &#181;L of 0.1% fast green (0.1% in 18 M&#937; deionized and distilled H2O [ddH2O]) for every 7 &#181;L of DNA mixture.</li>
		<li>For co-electroporation of multiple plasmids, mix plasmids in a 1:1 ratio. The final DNA concentration should be 5-8 &#181;g/&#181;L.</li>
	</ol>
	</li>
	<li>Fill Injection Pipette
	<ol>
		<li>Pull pipettes on a micropipette puller. Fill the pipette with 1-2 &#181;L plasmids using a 28G syringe needle.</li>
		<li>Break pipette tip to 10 - 20 &#181;m using forceps. Use a microscope to assist in determining the size of the broken pipette tip. Attach the pipette to the pipette holder of the picospritzer.</li>
	</ol>
	</li>
</ol>
3. Windowing
NOTE: The windowing procedure has been published before13. Please see reference 13 for additional visual information.
<ol>
	<li>Wipe all instruments and the work area with 70% ethanol.</li>
	<li>Take the desired number of set eggs out of the incubator (between 6-10), leave at room temperature, and individually swab eggs with 70% ethanol again. Eggs remain viable for at least 2 h after incubator removal.</li>
	<li>Place the egg on top of a light illuminator to identify embryo positioning. Circle the center of the dark area (i.e., embryo) with a pencil.</li>
	<li>Using a 19G needle, poke a hole in the blunt end of the egg.</li>
	<li>Poke another hole near the drawn circle on the pointed side of the egg. Remove a small piece of the outer eggshell (about 16 mm2) with the 19G needle and then remove a small piece of the inner eggshell (about 8 mm2) with forceps.</li>
	<li>With a 3 mL syringe fitted with a 19G needle, withdraw 1.5-2.5 mL of albumin from the egg through the blunt-end hole. Be sure to angle the needle down at 45&#176;.</li>
	<li>Cover the blunt-end hole with a small piece of tape (1 cm x 1 cm). Cover the drawn circle and the second hole with tape (2.5 cm x 4 cm).</li>
	<li>With curved scissors (cutting edge = 2.5 cm), cut a window within the circle. Scissors should be parallel to the eggshell and the diameter of the window should be less than 2 cm.</li>
</ol>
4. Plasmid Injection
<ol>
	<li>Turn on the picospritzer. Set the pressure to 18 psi and the duration to 5 &#181;s. Turn on the air tank and lamp for the dissection microscope.</li>
	<li>Make 30 mL stock solution of a 1:10 dilution of store-bought Indian ink mixed in sterile phosphate-buffered saline (PBS). Store at 4 &#176;C. Injection of Indian ink is an important step to get a better visualization of the chicken embryo.
	<ol>
		<li>Fill a 1 mL syringe with the diluted Indian ink solution. Attach a 27G needle and expel any bubbles that are present in the syringe.</li>
		<li>Insert needle just under the yolk membrane ~2 - 3 mm from the embryo and inject ~0.2 mL of Indian ink gently under the embryo. Do not inject more than 0.5 mL of the Indian ink as it may decrease embryo survival.</li>
	</ol>
	</li>
	<li>Place the windowed egg on an egg holder under the dissection microscope. Use the highest magnification for best visualization of plasmid injection site.</li>
	<li>Using forceps, remove the membrane over the injection area. Auditory brainstem regions of interest (i.e., NM and NL) arise from Rhombomeres 5 and 6 (R5/R6). R5/R6 are located adjacent to the otocysts (an embryonic structure in vertebrates that develops into the inner ear) that serve as landmarks for R5/R6 plasmid injections.</li>
	<li>Lower the DNA-filled pipette into the neural tube overlying the R5/R6 region and between otocysts using the micromanipulator. A schematic of ideal placement is shown in Figure 1A.</li>
	<li>Apply air pressure (18 psi, 5 &#181;s duration) from the picospritzer to eject DNA in the neural tube.</li>
</ol>
5. Electroporation
<ol>
	<li>Turn on the current/voltage stimulator.</li>
	<li>Fill a 3 mL syringe with PBS and attach the syringe filter. Add 1-2 drops of PBS onto the embryo.</li>
	<li>Lower the bipolar electrode to the embryo using the micromanipulator with the negative electrode above the injection site (medial) and the positive electrode lateral to the R5/R6 (Figure 1A). Avoid direct contact with the embryo. Use bipolar electrodes where the electrode material is platinum iridium. Adjust the spacing between the tips to 0.5 mm with forceps.</li>
	<li>Using a current/voltage stimulator, pulse 20 times at 50 V for 1 ms at 1 s intervals.</li>
	<li>After electroporation, add one drop of PBS over the exposed area. Gently remove the electrode and clean it with a laboratory tissue and 70% ethanol. Close the window exposing the embryo with tape.</li>
	<li>Label eggshell with the injected plasmid type and the hatch date.</li>
	<li>Place eggs back into incubator with window side up. Incubate at 38 &#176;C with 50% humidity until desired developmental stage is reached.</li>
	<li>If a tet-on vector is electroporated for temporal control of gene expression, apply Doxycycline (Dox) every 24 h to induce gene expression (see Section 6).</li>
</ol>
6. Tet-on System for Temporal Control of Gene Expression
<ol>
	<li>Use the following plasmids: 
	pCAGGS-T2TP: a transposase expressing plasmid. 
	pT2K-CAGGS-rtTA-M2: a plasmid that stably expresses the Dox-binding protein. 
	pT2K-BI-TRE-EGFP: a plasmid that contains a bidirectional tet-on promoter (TRE)-driving expression of the EGFP reporter and a second gene of interest. 
	NOTE: The above three plasmids need to be co-electroporated in a 1:1:1 ratio.</li>
	<li>Stock solution preparation:
	<ol>
		<li>Dissolve Dox in sterile PBS to produce a 1 mg/mL stock solution. Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Dox application:
	<ol>
		<li>To induce the expression of the gene of interest during certain developmental stages, apply Dox every 24 h.</li>
		<li>When applying Dox, take the egg out, open the window, pipette 50 &#181;L Dox onto the chorioallointoic membrane, close the window and put the egg back into the incubator.</li>
	</ol>
	</li>
</ol>
7. Preparation of Dissecting Area for Brainstem Slices
NOTE: The following sections (7 - 9) and procedures have been published before14. Please see these references for additional visual information.
<ol>
	<li>Prepare an agar solution (40 mg/mL agarose, i.e. 4% in ddH2O). Pour agar solution into a Petri dish and allow it to solidify at room temperature. Store covered agar plate in the refrigerator for future use during brainstem slicing of tissue.</li>
	<li>Continually bubble artificial cerebral spinal fluid (ACSF) with 95% O2 and 5% CO2 (pH 7.2-7.4 and osmolarity: 295-310 mOsm/L).</li>
	<li>Clean the working area and vibratome with 70% EtOH and rinse the blade with distilled water.</li>
	<li>Place a clean fluid absorption pad on the working area with appropriate dissecting tools.</li>
	<li>Take the agar plate out of the refrigerator, cut a small agar cube (10 mm x 10 mm x 8 mm). Glue the agar block to the stage of a vibratome-slicing chamber using commercially available superglue.</li>
</ol>
8. Isolation of Chicken Auditory Brainstem
<ol>
	<li>Open the egg at taped window site. Puncture the membrane sac with a scalpel and remove the head of the chicken embryo.</li>
	<li>Decapitate the chicken with sharp scissors.</li>
	<li>Place the razor blade tip slightly posterior to the eyes. Incise through the skull at the rostral-to-caudal midline. Apply slight pressure depending on the age of the embryo. Older embryos require more pressure.</li>
	<li>Gently push aside skin and feathers to expose skull and verify midline cut.</li>
	<li>Applying strong pressure, slice the rostral portion of skull with a razor blade. Immediately place blade posterior to eyes and cut through entire skull and brain tissue.</li>
	<li>Make midline-to-lateral incisions with scissors in the skull&#34;s caudal region, slightly anterior to neck muscles on both sides of the head. Expose brain and cerebellum by pulling away skull and excess tissue.</li>
	<li>Cut tissue attached to the brainstem with scissors and remove the brainstem, which freely detaches from the skull.</li>
</ol>
9. Preparation of Brainstem Slices for In Vivo Electrophysiology or Imaging
<ol>
	<li>Pin down brainstem through the optic tecta.</li>
	<li>Remove the cerebellum by cutting peduncles with scissors, exposing the floor of the fourth ventricle.</li>
	<li>Using tweezers, remove any membranous tissue and blood vessels from the brainstem surface.</li>
	<li>To block the brainstem, make a horizontal cut at the most rostral end of the floor of the fourth ventricle, just caudal to the cortex. Make lateral cuts perpendicular through the optic tecta to isolate brainstem.</li>
	<li>Place a small amount of super glue on the vibratome stage directly in front of the agar block.</li>
	<li>Lift the brainstem at the spinal cord using tweezers and place it on the super glue with rostral side down and dorsal side towards the vibratome blade. Remove excess glue with a laboratory tissue or filter paper.</li>
	<li>Pour oxygenated ACSF into the vibratome stage. Set the vibratome blade at a 20-22&#176; angle. Start vibratome at maximum-amplitude oscillation. Move the stage up towards the blade so the top of the tissue is parallel with the blade.</li>
	<li>Rapidly move blade towards brainstem tissue. Slow down the blade considerably prior to the blade&#34;s contact with tissue.</li>
	<li>Slice tissue with the slowest possible forward speed. Once the blade is through the entire coronal tissue section, gently remove the slice using a transfer pipette.</li>
	<li>Lower the stage 200-300 &#181;m and slice again. Repeat until anatomical landmarks of auditory nuclei become visible, e.g., the distinct dorsal/ventral neuropil region of NL and the medial location of NM relative to NL.</li>
	<li>Carefully maintain slices in ACFS. This can be done at room temperature (22&#176; C) or near physiological conditions (~41&#176; C) using a warm water bath. Note the order of slicing. The first slice corresponds to the most caudal region of tissue and the last slice corresponds to the most rostral region. This roughly represents the tonotopic organization of the auditory brainstem, from low- to high-frequency encoding regions, respectively.</li>
</ol>

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The authors have nothing to disclose.

In ovo electroporation is a method of expressing or knocking down genes of interest in order to analyze in vivo gene function8,9. In the chicken embryo, it is an innovative method for expressing plasmid-encoded genes into different auditory brainstem regions8. To ensure optimal expression, several critical steps are required. First, only inject embryos whose otocysts are clearly visible. If otocysts are not visible the em...

Fast neural encoding of sound is essential for normal auditory functions. These include sound localization abilities1, speech in noise discrimination2, and the comprehension of other behaviorally relevant communication signals3. Analogous neurons located in the auditory brainstem of both avians and mammals are highly specialized for fast neural encoding4. These include the chicken nucleus magnocellularis (NM), the nucleus laminaris (NL) and their mammalian analogs, the anteroventral cochlear nucleus (AVCN) and the medial superior olive (MSO), respectively5. However, developmental mechanisms regulating fast neural encoding are poorly understood in the auditory brainstem. Therefore, it is advantageous to study specific genes that are responsible for fast neural encoding in order to better understand their expression, regulation and function in auditory development.
The developing chicken embryo is an effective and well-established research tool to study basic biological questions of auditory system development6,7. Recent molecular advances have addressed these biological questions in the developing chicken embryo by expressing or knocking down genes of interest in order to analyze in vivo gene function8,9. Investigating the regulatory role of specific genes is a significant advancement in understanding pathologies associated with auditory deficits. Here, we present in ovo electroporation of plasmid-encoded genes into the chicken auditory brainstem where fast neural encoding of sound occurs10. By targeting auditory neural progenitor regions associated with rhombomeres 5 and 611,12 (R5/R6), we show spatial control of plasmid transfection in NM and NL. In addition, we show temporal regulation of expression 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)8.

<table><tbody><tr><td>Fertilized white leghorn chicken eggs</td><td>Sunnyside Inc. (Beaver Dam, WI)</td><td></td><td></td></tr><tr><td>Picospritzer</td><td>Parker Hannifin</td><td>052-0500-900</td><td>Picospritzer III, single or dual channel</td></tr><tr><td>Current/voltage stimulator</td><td>Grass Technologies</td><td>SD9</td><td>SD9</td></tr><tr><td>Microfil syringe needles</td><td>World Precision Instruments</td><td>MF28G67-5</td><td>28 Gauge, 67 mm Long, (Pack of 5)</td></tr><tr><td>Electrode holder</td><td>Warner Instruments</td><td>64-1280</td><td>MP Series: Non-Electrical Pressure Applications</td></tr><tr><td>Stimulating microelectrode</td><td>FHC</td><td>PBSA1075</td><td>PBSA1075</td></tr><tr><td>Air tank/regulator</td><td>NU Laboratory Services</td><td></td><td>Air dry 300 CF</td></tr><tr><td>Fast green</td><td>Sigma Aldrich</td><td>F7258-25G</td><td>F7258-25G</td></tr><tr><td>Clear plastic tape</td><td>Scotch</td><td>191</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma Aldrich</td><td>D9891-1G</td><td></td></tr><tr><td>Egg refrigerator</td><td>Vissani Wine Refrigerator</td><td></td><td>13.3 - 16.1 &amp;deg;C (56-61&amp;deg; F)</td></tr><tr><td>Incubator</td><td>Hova-Bator</td><td></td><td>38 &amp;deg; C (100 &amp;deg; F), ~50% humidity</td></tr><tr><td>Dissection scope</td><td>Zeiss</td><td>4.35E+15</td><td>SteREO Discovery, V8 Microscope, 50.4X</td></tr><tr><td>Cold-light source</td><td>Zeiss</td><td>4.36E+15</td><td>CL6000 LED</td></tr><tr><td>Micromanipulators</td><td>Narishige Japan</td><td>Model: MM-3</td><td>2 Micromanipulators</td></tr><tr><td>Capillary tubes</td><td>Sutter Instrument</td><td>BF150-86-10</td><td>Thick-walled borosilicate (dimensions)</td></tr><tr><td>Syringes</td><td></td><td></td><td>1 mL, 3 mL</td></tr><tr><td>Needles</td><td>BD Precision Glide&amp;nbsp;</td><td></td><td>27 G x 1 1/4, 19 G x 1 1/2</td></tr><tr><td>Forceps</td><td>Stoelting</td><td></td><td>No. 5 Super Fine Dumont</td></tr><tr><td>Egg holder</td><td>Custom Made</td><td></td><td>Clay base works as well</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td></td><td>Model P-97</td></tr><tr><td>Syringe filter</td><td>Ultra Cruz</td><td>sc-358811</td><td>PVDF 0.22 &amp;mu;m</td></tr></tbody></table>

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.

We show here that in ovo electroporation permits gene expression in a normally developing biological system. Plasmid-encoded genes are focally injected into the neural tube overlying R5/R6. A schematic example of the electrode and pipette placements relative to important anatomical markers is shown in Figure 1A. The correct location of plasmid injection is confirmed 24 h after electroporation and shown in Figure 1B. The targeted injection of plas...

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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.

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 ...

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Research

JoVE Journal

Developmental Biology

8.4K Views.  Northwestern University.  Auditieve hersenstam neuronen van avians en zoogdieren zijn gespecialiseerd in snelle neurale codering, een fundamenteel proces voor normale gehoorfuncties. Deze neuronen ontstaan ​​uit duidelijke precursoren van embryonale hindbrain. We presenteren technieken die elektroporatie gebruiken om genen uit te drukken in het achterhoofd van kippenembryo's om de genfunctie tijdens de auditieve ontwikkeling te bestuderen. 

Video: In Ovo Electroporation in the Chicken Auditory Brainstem

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 ...

Neuroscience

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 Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biology

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

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.

In Ovo Electroporation in the Chicken Auditory Brainstem

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