This work is supported by the NIH/NIDCD R01 DC017167 grant.&#160;We thank Kristine McLellan for providing editorial comments on an earlier version of the manuscript.

All procedures were approved by Northwestern University Institutional Animal Care and Use Committees (IACUC) and were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The protocols for dissection and preparation of brainstem tissue are in adherence with previous protocols5,13.
1. Egg handling
<ol>
	<li>Purchase fertilized eggs (Gallus gallus domesticus) from an IACUC-approved local animal supplier.</li>
	<li>Store the eggs immediately upon arrival in a refrigerator at 14 &#176;C and incubate within 5 days. 
	NOTE: Embryo viability decreases considerably after 1 week.</li>
	<li>Sterilize the eggs with 70% ethanol before incubation at 38 &#177; 1 &#176;C and ~50% humidity.</li>
</ol>
2. Artificial cerebral-spinal fluid (ACSF) composition and preparation
<ol>
	<li>Mix the following chemicals in 1 L of 18.2 M&#8486;cm dH2O to create a 10x ACSF stock solution: NaCl (sodium chloride) 130 mM, NaHCO3 (sodium bicarbonate) 26 mM, KCl (potassium chloride) 2.5 mM, NaH2PO4 (sodium dihydrogen phosphate) 1.25 mM, dextrose (D-(+)-glucose) 10 mM. Keep the stock solution in the refrigerator.</li>
	<li>Prepare MgCl2 (magnesium chloride) 1 M and CaCl2 (calcium chloride) 1 M solutions separately in 18.2 M&#8486;cm dH2O and store in the refrigerator.</li>
	<li>Immediately before use, dilute 10x ACSF to 1x and bubble continuously with 95% O2/5% CO2 for 15-20 min and add MgCl2 and CaCl2. To prepare ACSF and dACSF (dissecting ACSF), adjust to a final concentration of Mg2+ 1 mM, Ca2+ 3 mM and Mg2+ 3 mM, Ca2+ 1 mM, respectively.</li>
	<li>Set the bubbling rate for the ACSF so that the pH is 7.2-7.4 with an osmolality between 300 and 310 mOsm/L. 
	&#8203;NOTE: Having the ACSF placed in an ice bath while bubbling is beneficial for maintaining a low solution temperature, which will support tissue structural integrity while dissecting.</li>
</ol>
3. Agarose (5%) block preparation
<ol>
	<li>Mix 5 g of agarose in 100 mL of dACSF. Use a 100 &#176;C water bath or microwave for 2-3 min, stirring every 30 s to prevent lumps until the agarose dissolves completely and starts bubbling.</li>
	<li>Pour the melted agarose in an empty Petri dish up to 5 mm thickness and keep at room temperature to set. After setting, seal the Petri dish using parafilm and store at 4 &#176;C.</li>
	<li>Cut the agarose into cubical blocks with a sharp blade and use them at the time of dissection.</li>
</ol>
4. Dissection protocol and isolating auditory brainstem
<ol>
	<li>Clean the dissection area using 70% ethyl alcohol solution spray.</li>
	<li>Glue the supporting or angled agarose block onto the vibratome tray.</li>
	<li>Choose eggs of the desired age (E20 and E21 in the present protocol). Handle and incubate the eggs following the protocols listed above as in step 1.</li>
	<li>Locate the air-filled space by placing the egg under a bright light (candling) and looking for this space on the larger or rounder side of the egg.</li>
	<li>Acclimate the eggs to room temperature, crack the shell over the air-filled space, and expose the membrane sac.</li>
	<li>Make a gentle incision in the sac to expose the beak.</li>
	<li>With a scalpel, gently pull the neck and head out of the egg.</li>
	<li>Quicky decapitate the head using sharp scissors.</li>
	<li>After decapitation, clean the head with ice-chilled dACSF to remove excess blood from the dissection pad.</li>
	<li>Hold the head steady in ice-chilled dACSF and make a rostro-caudal incision. Start the incision behind and between the eyes and follow the length of the harvested neck. 
	NOTE: Younger embryos may require less pressure while making the incision.</li>
	<li>Separate the skin to expose the skull.</li>
	<li>Cut the skull behind the eye in a midline to lateral direction. Do this for both hemispheres. 
	NOTE: This step helps separate the rostral portion of the skull from the attached brain while keeping the brain tissue intact5.</li>
	<li>Slice the rostral portion of the skull. Place the blade behind the eyes and make a quick cut. 
	NOTE: Effort may be required to cut the attached skull cleanly.</li>
	<li>Immerse the head in a dish of cold dACSF.</li>
	<li>Utilizing a small pair of scissors, make midline to lateral incisions in the caudal region of the skull to try and separate the brain from the skull without causing tissue damage.</li>
	<li>Gently expose the brainstem and cerebellum. Retract the dorsal area of the entire skull, remove the brainstem carefully, and expose it with the help of a fine paintbrush with mild sledding. Use curved forceps to clean the brainstem from connecting tissue and blood vessels. Pay extra attention to the 8th cranial nerve area and be sure to leave a short length of intact nerve fibers on both sides.</li>
	<li>Separate the brainstem from the cerebellum by cutting the peduncles and removing the blood vessels carefully. Trim the brainstem of additional blood vessels. 
	NOTE: Ensure that the whole procedure is performed in ice-chilled dACSF continuously bubbled with carboxygen (95% O2/5% CO2).</li>
</ol>
5. Vibratome slicing
NOTE: In the following steps, the back of the tissue must be supported with a cubical piece of agarose.
<ol>
	<li>Place the vibratome blade along the horizontal axis and glue the brainstem on a slicing tray. Glue the rostral side, keeping the rostral-caudal axis vertical for coronal slices.
	<ol>
		<li>Keep the lateral-medial axis vertical for sagittal slices.</li>
		<li>Glue the ventral side, keeping the dorsal-ventral axis vertical for horizontal slices.</li>
	</ol>
	</li>
	<li>To achieve the acute angular sagittal-horizontal plane, glue the brainstem&#39;s ventral side, keeping the ventral-dorsal axis vertical on the agarose block&#39;s hypotenuse surface, which is cut at a 45&#176; angle. Glue the opposite surface of the agarose block facing the slicing tray and keep the rostral-caudal axis parallel to the blade edge.</li>
</ol>
6. Handling fragile or large pieces of brainstem tissue
<ol>
	<li>In an alternative approach to step 5, immerse the isolated brainstem in 4% low melting point (LMP) agarose at ~40 &#176;C in a 35 mm x 10 mm Petri dish.
	<ol>
		<li>After pouring agarose on the immersed brainstem, place the Petri dish on ice to solidify. Cut the cubical agarose block with embedded brainstem using a sharp razor blade.</li>
	</ol>
	</li>
	<li>Glue the LMP agarose block on its rostral side, keeping the brainstem&#39;s rostral-caudal axis vertical.
	<ol>
		<li>Take coronal slices until the NM region can be visualized.</li>
		<li>Remove the agarose block from the glue with a sharp blade. To spot the nuclei, gently place 0.5 &#181;L of dye (toluidine blue or orange G) on the NM with a fine needle.</li>
		<li>Remount this block on the slicing tray for sagittal or horizontal slices and identify the nuclei with respect to the stained region.</li>
	</ol>
	</li>
	<li>For the best performance, set the vibratome slicing speed at 4 - 5 (~30 &#177; 4 mm/min), vibrating frequency at 85-87 Hz, and slicing amplitude at 4-6 (~1 &#177; 0.2 mm).</li>
	<li>After brainstem sectioning, place the 200-300 &#181;m sequentially collected slices in a commercially available slice chamber to equilibrate for 1 h at room temperature in ACSF, continuously bubbled with a mixture of 95% O2/5% CO2 (pH 7.2-7.4, osmolarity 300-310 mOsm/L). In these conditions, slices remain viable up to 5-6 h.</li>
</ol>
7. Electrophysiology: patch clamp procedure
<ol>
	<li>Transfer a brainstem slice to the recording chamber with continuous perfusion of carboxygenated ACSF ~1.5 &#177; 0.5 mL/min.</li>
	<li>Pull patch pipettes with a micropipette puller of tip diameter 1-2 &#181;m and resistance in the range of 3-6 M&#8486;.
	<ol>
		<li>Fill pipettes with a K-Gluconate-based internal solution (for current-clamp recording).</li>
	</ol>
	</li>
	<li>To test the neuronal properties across the different tonotopic regions within a slice, locate neurons at either end of the slice plane and approach with the recording electrode.</li>
	<li>Maintain positive air pressure at the pipette tip while approaching a neuron.</li>
	<li>Move towards the soma until an indentation is visualized on the neuron. Perform the next two steps quickly.</li>
	<li>Make a gigaohm (1 G&#8486;) seal by releasing positive air pressure.</li>
	<li>Keep the amplifier setting in voltage-clamp mode and correct the pipette offset as zero pA. Run a seal test (10 mV test pulse at 100 Hz). Apply negative air-pressure to rupture a small patch of the neuronal membrane.</li>
	<li>To test the active intrinsic properties of auditory neurons, apply hyperpolarizing and depolarizing somatic current injections. 
	NOTE: Examples of this procedure can be visualized in Supplementary Video S1,&#160;S2. Details of this procedure are provided in the video legends.</li>
</ol>

<ol>
	<li>Rubel, E. W., Parks, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+development+of+brain+stem+auditory+nuclei+of+the+chicken:+tonotopic+organization+of+n.+magnocellularis+and+n.+laminaris.">Organization and development of brain stem auditory nuclei of the chicken: tonotopic organization of n. magnocellularis and n. laminaris.</a> Journal of Comparative Neurology. 164 (4), 411-433 (1975).</li><li>Rubel, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+development+of+brain+stem+auditory+nuclei+of+the+chicken:+ontogeny+of+n.+magnocellularis+and+n.+laminaris.">Organization and development of brain stem auditory nuclei of the chicken: ontogeny of n. magnocellularis and n. laminaris.</a> Journal of Comparative Neurology. 166 (4), 469-489 (1976).</li><li>Shao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+synaptic+activity+in+chick+vestibular+nucleus+neurons+during+the+perinatal+period.">Spontaneous synaptic activity in chick vestibular nucleus neurons during the perinatal period.</a> Neuroscience. 127 (1), 81-90 (2004).</li><li>Fukui, I., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonotopic+gradients+of+membrane+and+synaptic+properties+for+neurons+of+the+chicken+nucleus+magnocellularis.">Tonotopic gradients of membrane and synaptic properties for neurons of the chicken nucleus magnocellularis.</a> Journal of Neuroscience. 24 (34), 7514-7523 (2004).</li><li>Sanchez, J. T., Seidl, A. H., Rubel, E. W., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+culture+of+chicken+auditory+brainstem+slices.">Preparation and culture of chicken auditory brainstem slices.</a> Journal of Visualized Experiments. (49), e2527 (2011).</li><li>Sanchez, J. T., Lu, Y., Fay, R. R., Popper, A. N., Cramer, K., Coffin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+signaling+in+the+auditory+brainstem.">Glutamate signaling in the auditory brainstem.</a> Auditory Development and Plasticity: Springer Handbook of Auditory Research. 64 (4), 75-108 (2017).</li><li>Parks, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+axosomatic+endings+in+an+avian+cochlear+nucleus:+nucleus+magnocellularis+of+the+chicken.">Morphology of axosomatic endings in an avian cochlear nucleus: nucleus magnocellularis of the chicken.</a> Journal of Comparative Neurology. 203 (3), 425-440 (1981).</li><li>Jhaveri, S., Morest, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+alterations+of+neuronal+architecture+in+nucleus+magnocellularis+of+the+developing+chicken:+a+Golgi+study.">Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: a Golgi study.</a> Neuroscience. 7 (4), 837-853 (1982).</li><li>Carr, C. E., Boudreau, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+projections+of+auditory+nerve+fibers+in+the+barn+owl.">Central projections of auditory nerve fibers in the barn owl.</a> Journal of Comparative Neurology. 314 (2), 306-318 (1991).</li><li>K&#246;ppl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+nerve+terminals+in+the+cochlear+nucleus+magnocellularis:+differences+between+low+and+high+frequencies.">Auditory nerve terminals in the cochlear nucleus magnocellularis: differences between low and high frequencies.</a> Journal of Comparative Neurology. 339 (3), 438-446 (1994).</li><li>Fukui, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+phase+information+at+low+sound+frequency+in+nucleus+magnocellularis+of+the+chicken.">Improvement of phase information at low sound frequency in nucleus magnocellularis of the chicken.</a> Journal of Neurophysiology. 96 (2), 633-641 (2006).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+FMRP+regulates+synaptogenesis+in+vivo+in+the+developing+cochlear+nucleus.">Postsynaptic FMRP regulates synaptogenesis in vivo in the developing cochlear nucleus.</a> Journal of Neuroscience. 38 (29), 6445-6460 (2018).</li><li>Lu, T., Cohen, A. L., Sanchez, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporation+in+the+chicken+auditory+brainstem.">In ovo electroporation in the chicken auditory brainstem.</a> Journal of Visualized Experiments. (124), e55628 (2017).</li><li>Hong, H., Sanchez, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Need+for+speed+and+precision:+structural+and+functional+specialization+in+the+cochlear+nucleus+of+the+avian+auditory+system.">Need for speed and precision: structural and functional specialization in the cochlear nucleus of the avian auditory system.</a> Journal of Experimental Neuroscience. (12), 1-16 (2018).</li><li>Hong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+intrinsic+properties+shape+functional+phenotype+of+low-frequency+neurons+in+the+auditory+brainstem.">Diverse intrinsic properties shape functional phenotype of low-frequency neurons in the auditory brainstem.</a> Frontiers in Cellular Neuroscience. 12, 1-24 (2018).</li><li>Wang, X., Hong, H., Brown, D. H., Sanchez, J. T., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+properties+in+the+low-frequency+region+of+the+chicken+cochlear+nucleus+magnocellularis.">Distinct neural properties in the low-frequency region of the chicken cochlear nucleus magnocellularis.</a> eNeuro. 4 (2), 1-26 (2017).</li><li>Tabor, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonotopic+organization+of+the+superior+olivary+nucleus+in+the+chicken+auditory+brainstem.">Tonotopic organization of the superior olivary nucleus in the chicken auditory brainstem.</a> Journal of Comparative Neurology. 520 (7), 1493-1508 (2012).</li><li>Lipovsek, M., Wingate, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+divergent+development+of+brainstem+vestibular+and+auditory+nuclei.">Conserved and divergent development of brainstem vestibular and auditory nuclei.</a> Elife. 7, 40232 (2018).</li><li>Passetto, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+analysis+of+the+AMPA-type+neurons+in+the+Deiter's+vestibular+complex+of+the+chick+brain.">Morphometric analysis of the AMPA-type neurons in the Deiter's vestibular complex of the chick brain.</a> Journal of Chemical Neuroanatomy. 35 (4), 334-345 (2008).</li><li>Curry, R. J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+properties+of+avian+interaural+level+difference+sound+localizing+neurons.">Intrinsic properties of avian interaural level difference sound localizing neurons.</a> Brain Research. 1752, 147258 (2021).</li><li>Aleksandrowicz, E., Herr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+euthanasia+and+short-term+anesthesia+of+the+chick+embryo.">Ethical euthanasia and short-term anesthesia of the chick embryo.</a> ALTEX - Alternatives to Animal Experimentation. 32 (2), 143-147 (2015).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+removal+of+hypothalamic+or+other+brain+nuclei+of+the+rat.">Isolated removal of hypothalamic or other brain nuclei of the rat.</a> Brain Research. 14 (59), 449-450 (1973).</li></ol>

All authors declare that the research was conducted without any commercial or financial interest and that they do not have any conflicts of interest.

Coronal sections of chicken embryonic brainstem tissue have allowed the study of relative individual iso-frequency lamina for decades1,2,5. However, the tonotopic (i.e., frequency) organization of the chicken auditory brainstem is topologically convoluted and may be more accessible in other anatomical axes depending on the specific research question. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variations and its development across larger auditory brainstem areas are somewhat limited by coronal sections. To overcome this limitation, this protocol describes approaches in the sagittal, horizontal, and horizontal/transverse planes to provide additional examples of auditory brainstem tissue that exhibit maximum tonotopic properties and gradients in an individual brainstem section.
Sagittal sections of auditory brainstem regions show that different tonotopic areas are distributed across a larger region within the slice compared to coronal sections (sagittal auditory area = ~300-600 &#181;m, coronal auditory area = ~200-350 &#181;m). For example, NM and NL regions were visualized over a larger area along the rostro-caudal axis in sagittal sections (e.g., Figure 2B), and the functional tonotopic gradient that runs along this anatomical axis was largely contained within a single sagittal slice. This was further confirmed with current-clamp recordings of intrinsic neuronal differences that vary along the rostral-caudal gradient as previously reported14,15 (e.g., Figure 3C,D). Future experiments that highlight anatomical and immunohistochemical properties along the tonotopic axis could further investigate known gradients of auditory properties within a single sagittal slice plane. These include, but are not limited to, MAP2 staining and potassium channel expression patterns, which are known gradients of dendritic architecture and intrinsic properties of NM and NL that have been previously shown in successive coronal sections16.
Horizontal sections of auditory brainstem regions show that the NM and NL are located toward the midline. A portion of auditory axonal fibers run diagonally or perpendicular to the horizontal plane (Figure 4). These fibers can be followed by making an acute angular slice 45&#176; to the sagittal plane. The resulting horizontal/transverse slices were larger than the sagittal or horizontal slices, and long axonal fibers coursed through the rostro-caudal axis for both ipsilateral and contralateral sides. Both NM and NL can be visualized in a larger diagonal region (~400-700 &#181;m) such that contralateral connections can be visualized along a lateral-medial axis. Additionally, the horizontal/transverse slice plane also shows how the auditory regions and the resulting tonotopic gradient make an angular turn (Figure 5). Angular exposure of contralateral connections in a larger area makes these slices more suitable for electrophysiological stimulation and microcircuitry studies than traditional coronal slices.
Additional advantages 
The formation of auditory microcircuits requires spatiotemporal coordination of cues that promote neuronal survival, synaptogenesis, axonal differentiation, dendritic architecture, and maturation. Thus, an alternative brainstem sections of the chicken embryo auditory microcircuit can be used for the following research topics: morphological organization of neurons in topographically different dimensions; organizing and mapping the connectomes of all auditory and vestibular nuclei; identification and characterization of the activity patterns of circuit constituents in iso-frequency and tonotopic planes; the topographical organization of excitatory versus inhibitory microcircuits and relations to specialized neuron populations (nuclei); spatial location of auditory nuclei neurons and its predictive CF17; systematic targeting of specific tonotopic neuronal types; tracking progenitor cells and their development into conserved nuclei; genetic lineage of cells to the evolution of neuronal circuits18; comparative brainstem anatomy between species; investigation of vestibular circuits like Deiter&#39;s vestibular complex (DC)19; and synchrony and cross talk between vestibular nuclei.
A multifaceted approach using different slice planes may help answer fundamental questions about unknown anatomical and biophysical properties of brainstem microcircuits. A good example is the relationship between major auditory nuclei (NM, NA, NL, and SON) and the vestibular nuclei, including the dorsal nucleus of the lateral lemniscus (LLDp), the semilunar nucleus (SLu)20, and the tangential nucleus (TN)3. However, this protocol and these slice-based studies do have some limitations.
Precautions and limitations 
Depending on the institution performing the experiments, ethical guidelines and the handling of chicken embryos may differ. While the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals permits rapid decapitation, there are alternative methods for chicken embryo euthanasia21. Early developing chicken embryo brainstem tissue is soft, and delicate compared to older embryos. It has several connections and blood vessels on the surface that need extra caution when removing them. Tissue should be kept in ice-cold dACSF and perfused with 95% O2/5% CO2 to increase the viability.
The sagittal slicing method is only useful for ipsilateral tonotopy. This slicing method provides larger slices than coronal slices, the handling of which could be precarious. However, one can trim the slices using cross needle methods described in detail elsewhere22. Using 4% LMP agarose block embedded brainstem can save delicate structures in slices, but care must be taken not to pour excessively hot agarose. Setting it quickly by placing the agarose-blocked brainstem in a chilled environment for ~1 min makes slices more viable for electrophysiological recordings.
Application of superglue in excess amounts can be toxic. It must be applied minimally, and surplus amounts should be washed immediately by changing out the dACSF. For acute angular (45&#176;) slices, cutting the angle of the agarose block is critical; one can use a mirror to see the front angle while cutting the agarose block with a sharp blade. Commercially available blades might have a wax coating which should be wiped off with alcohol and dried before use. Optimization is required for the vibratome cutting speed and frequency as axonal fiber tufts are harder than cortical or matrix tissue. Keeping a high amplitude and using chilled dissection solution may prevent tissue damage.
All solutions should be prepared fresh, and Ca2+ and Mg2+ should be added to the ACSF after bubbling 95% O2/5% CO2 . Otherwise, there may be precipitation of Ca2+. A paintbrush should be used to handle the slices gently within the vibratome. Keep the total slicing time under 15 min if possible. A glass Pasteur pipette can be used to maneuver brainstem slices.
Do not use detergent or corrosive washing agents for glassware and equipment that contact the slices used in electrophysiology. The images taken represent the appearance of 200-300 &#181;M thick tissue under differential interference contrast (DIC) optics. The visual quality will be poorer than immunohistochemistry or electron microscopy, but it accurately reflects what an experimenter will see when performing electrophysiological recordings.
Studies pertaining to the early development of microcircuits along an alternative anatomical axis, whether they are dorsal-ventral, rostral-caudal, or ipsilateral-contralateral, are limited in the chicken auditory brainstem. One reason for this is because the role of transcriptional codes and regulation of tonotopic development in the brainstem is still not fully understood. Functional phenomena such as top-down modulation and spontaneous activity are often lost when observing activity in vitro. However, in vivo research is complemented by specific and direct single neuron recordings only possible in these slice conditions. The refinement of obtaining brainstem tissue along different orientations could provide insightful information about the development and complexity of tonotopic gradients in the chicken auditory brainstem microcircuitry.

The chicken embryo is a valuable research model to study basic biological questions in numerous and diverse scientific areas including cell biology, immunology, pathology, and developmental neurobiology. The microcircuitry of the chicken auditory brainstem is an excellent example of a highly specialized circuit that can be understood in terms of auditory morphology and physiology. For example, Rubel and Parks (1975) first described the tonotopic orientation (i.e., frequency gradient) of the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL) as a linear function across the axis of the nuclei, oriented ~30&#176; with respect to the sagittal plane. Individual neurons in NM and NL encode their best sound frequency-known as their characteristic frequency (CF)-along the rostral-medial plane to the caudo-lateral region. High-frequency-sensitive neurons are in the rostral-medial region and low-frequency-sensitive neurons are located caudo-laterally. As such, traditional dissection methods of auditory brainstem tissue to study tonotopic properties have utilized successive coronal slices. Indeed, auditory microcircuits of developing chicken embryos have been established as a model system for studying signal processing of tonotopic auditory functions through successive caudal-to-rostral coronal plane brainstem slices for decades1,2,3,4,5,6.
However, the tonotopic organization of NM and NL is topologically and morphologically convoluted. Auditory nerve inputs are distributed such that high CF inputs terminate in endbulb-like structures that cover at least one-quarter of an adendritic NM cell&#39;s somatic circumference. Conversely, low CF inputs are not organized with end bulb-like terminals but with multiple bouton synapses on dendrites of NM neurons. Middle CF inputs terminate as both end bulb and bouton-like synapses4,7,8,9,10,11,12. In NL, the highly stereotyped dendritic gradient is evident not only in dendritic length but also in dendritic width. This unique dendritic gradient closely conforms to the tonotopic axis. The dendrites undergo an 11-fold increase in length and fivefold increase in width from high- to low-CF neurons, respectively6. To overcome such convoluted distributions of these nuclei in coronal slices, this protocol describes dissection approaches in the sagittal, horizontal, and horizontal/transverse planes. These slicing techniques provide examples of auditory brainstem tissue that exhibit maximum tonotopic properties in an individual slice plane.

<table><tbody><tr><td>Adobe photoshop 2021</td><td>Adobe</td><td></td><td></td></tr><tr><td>Anti-vibration table 30&quot;x 36&quot; - OTMC - 63533</td><td>TMC</td><td></td><td></td></tr><tr><td>Cell sens standard software</td><td>OLYMPUS</td><td></td><td></td></tr><tr><td>Digidata 1440A</td><td>MOLECULAR DEVICES</td><td></td><td></td></tr><tr><td>Digital amplifier&amp;nbsp; multiclamp 700B</td><td>MOLECULAR DEVICES</td><td></td><td></td></tr><tr><td>DSK line-up linearslicer pro7</td><td>TED PELLA, INC</td><td></td><td></td></tr><tr><td>Micromanipulator MPC-385 / OSI-MPC-385-2</td><td>OLYMPUS AMERICA INC</td><td></td><td></td></tr><tr><td>Micropipette puller P-97</td><td>SUTTER INSTRUMENTS</td><td></td><td></td></tr><tr><td>Microscope BX51W1</td><td>OLYMPUS AMERICA INC</td><td></td><td></td></tr><tr><td>MS ICE software</td><td>Microsoft Corporation</td><td></td><td></td></tr><tr><td>Ohaus balance model AV212</td><td>Ohaus Adventurer</td><td></td><td></td></tr><tr><td>Olympus DPSI0 /DPS80 camera</td><td>OLYMPUS</td><td></td><td></td></tr><tr><td>pClamp and Axoclamp data Acquisition Softwares</td><td>MOLECULAR DEVICES</td><td></td><td></td></tr><tr><td>pH meter lab 850 benchtop</td><td>SCHOTT INSTRUMENTS</td><td></td><td></td></tr><tr><td>Sharp stainless blade</td><td>Dorco/Personna</td><td></td><td></td></tr><tr><td>Vapor pressure osmometer model 5600</td><td>WESCOR INC</td><td></td><td></td></tr><tr><td>Water purification systems Smart2pure 6UV/UF</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Chemicals- list&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agrose Low melt IB70051</td><td>IBI SCIENTIFIC</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (Calcium Chloride)</td><td>ACROS organics</td><td></td><td></td></tr><tr><td>Cynergy instant adhesive CA6001</td><td>Resinlab</td><td></td><td></td></tr><tr><td>Dextrose (D-(+)-glucose)</td><td>VWR Life Science</td><td></td><td></td></tr><tr><td>Ethyl alcohol</td><td>IBI SCIENTIFIC</td><td></td><td></td></tr><tr><td>KCl (Potassium Chloride)</td><td>Amresco.Inc</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;(Magnesium Chloride)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>NaCl (Sodium Chloride)</td><td>Amresco.Inc</td><td></td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (Sodium Dihydrogen Phosphate)</td><td>Amresco.Inc</td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt; (Sodium Bicarbonate)</td><td>Amresco.Inc</td><td></td><td></td></tr></tbody></table>

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.

All brainstem slices shown here were acquired from brainstem tissue (~200-300 &#956;m) and imaged using a 5x objective and differential interference contrast (DIC) optics. The camera was mounted on the dissecting microscope and connected to a computer with image acquisition software (see Table of Materials). Satellite inset for these figures (right panels) were imaged using a 60x magnification water immersion objective. Care was taken to ensure that all areas of the brainstem slice were equally magnified while obtaining digital images. Photographs were taken at optimal brightness and focus. The digital images of brainstem slices were stitched in a planar fashion based on overlapping area and imported to a desktop computer for further adjustments of brightness, contrast, and grayscale. The basic microcircuits of the chicken auditory brainstem were identified according to previous work1,2,5,13. Under the microscope (5x objective), auditory nuclei were identified as the area adjacent to the heavily myelinated nerve fibers coursing around each nucleus both ipsilaterally and contralaterally along the dorsal regions of the slice.
Figure 1 shows the traditional coronal sections of brainstem tissue (200-300 &#181;m) from an E21 chicken embryo. The four coronal slices shown here represent the relative iso-frequency regions of the auditory brainstem nuclei, from the lowest-CF auditory region (Figure 1A, caudo-lateral) progressing to the highest-CF auditory region (Figure 1D, rostral-medial). For all four coronal slices in Figure 1A-D, magnified regions of labeled NM and NL are shown in the middle column and magnified (60x objective) on the right sight of the figure panel (a and b, respectively, in satellite insets). The arrow in Figure 1A,B shows the input of auditory nerve fibers, and the arrowhead shows bifurcation of NM axons on the left of the slice. Figure 1C shows another avian cochlear nucleus structure known as nucleus angularis (NA, arrow on left and arrowhead on right). The two most rostral coronal slices show the superior olivary nucleus (SON) located along the ventral-lateral region of the coronal slice (Figure 1C,D, white dashed circles).
Figure 2 shows sagittal sections of brainstem tissue (200-300 &#181;m) from a E21 chicken embryo. For all three sagittal slices (Figure 2A-C), magnified regions of labeled NM and NL are shown in the middle column and magnified (60x objective) on the right sight of the figure panel (a and b, respectively in satellite images). NM and NL were identified where the auditory nerve fibers (Figure 2A,&#160;middle arrow) entered the cluster of neurons observed at higher magnification (Figure 2A, middle, small, white dashed circles and arrowheads) and highlights the starting point of the auditory region (Figure 2A, left, large, white dashed circle and arrowhead). SON was identified in the rostro-lateral region of the most lateral slice (Figure 2A, small, white, dashed circle). Figure 2B shows extended tonotopic regions that contain both relatively low- and high-CF auditory regions from NM and NL along the rostral-caudal axis (white outlined regions, see also satellite inset ). Figure 2C shows the&#160;ipsilateral and contralateral axonal tufts in the most medial slice and the endpoint of the auditory region (left and middle arrows).The orientation of the slices shown here contrast with the traditional orientation of slices as seen in Figure 1 (i.e., coronal). This was performed to display the orientation that best accommodates the approach of a glass pipette required for electrophysiological recordings.
To confirm that a large region of the tonotopic axis was represented in Figure 2B, current-clamp electrophysiology recordings were performed from NM neurons. Figure 3 shows functional similarities and differences of mature (E21) NM neurons recorded from a coronal slice (Figure 3A,B) and a sagittal slice (Figure 3C,D, Supplementary Video S1, S2). Two NM neurons were selected from the medial and lateral ends of a coronal slice (similar to the slice shown in Figure 1B), and two NM neurons were selected from the rostral and caudal ends of NM in a sagittal slice (as in the slice shown in Figure 2B). Figure 3A,B shows similar electrophysiological response properties to somatic current injections (&#8722;100 pA to +200 pA, +10 pA increments, 100 ms duration). The firing pattern of these two NM neurons exhibits subtle differences in this slice plane, indicating relative iso-frequency lamina for mid-frequency NM neurons. Figure 3C,D show that the firing patterns have substantive differences across the rostral-caudal axis, indicating a relatively higher tonotopic gradient from a low frequency NM neuron (Figure 3C) to a high-frequency NM neuron (Figure 3D). Both neurons presented&#160;with their stereotypical firing patterns as previously reported14,15.
Figure 4 shows horizontal sections of brainstem tissue (200-300 &#181;m) of an E21 chicken embryo. For both horizontal slices (Figure 4A,B), magnified regions of labeled NM and NL are shown in the middle column and magnified (60x objective) on the right sight of the figure panel (a and b, respectively, in satellite insets). In the horizontal slices, NM and NL were identified toward the midline and neurons were spread along the lateral-medial axis (Figure 4A,B,&#160;middle, white, dashed outline regions). The magnified images show the large extent of the tonotopic gradient. Low-frequency neurons are in the caudo-lateral regions and high-frequency neurons are in the rostral-medial areas (Figure 4A,B, right, satellites). The fibers running through the midline along the rostral-caudal axis show the contralateral connections of the auditory nuclei, but the organization of these fibers is not in a simple plane. However, acute angular slices from a horizontal/transverse section can follow these axonal fibers towards the sagittal plane. Slices of 200-300 &#181;M thick brainstem tissue at an acute angle (45&#176;)&#160;from a horizontal plane are shown in Figure 5. Auditory brainstem nuclei can be seen across a large diagonal spread starting from the most lateral slice and ending at the most medial slice (Figure 5A-C, labeled middle panels, white outlined area). Moreover, the angular orientation of NM and NL regions can also be visualized in successive asymmetrical slices (Figure 5A-C, labeled middle panels, white, dashed outlined area). Magnified images (60x objective) show the tonotopic axis of the auditory nuclei as it courses along the rostral-medial to caudo-lateral axis (Figure 5A-C, right, satellite inset). The orientation of slices in Figure 5 is similar to that in Figure 2. They contrast the traditional presentation of images but are more suitable for electrophysiological experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63476/63476fig01.jpg" /> 
Figure 1: Representative coronal serial sections of the brainstem. (A-D) Left: slices from caudal to rostral axis,&#160;the auditory nuclei and connecting fibers marked with a white dashed circle. The middle insert is a larger view of the auditory region, where nuclei are shown within white dashed circles a: NM and b: NL. Arrows show auditory nerve afferent fibers, and arrowhead shows NM axon bifurcation in A,B. Arrow shows NA in C. Lateral white dashed circle shows SON in C,D. Right: satellite insert shows these nuclei at 60x objective: a: NM and b: NL. Abbreviations: NM = nucleus magnocellularis; NL = nucleus laminaris; NA = nucleus angularis; SON = superior olivary nucleus; LF = relatively low-frequency neurons; MF = medium-frequency neurons; HF = high-frequency neurons; D = dorsal; L = lateral; V = ventral. <a href="https://www.jove.com/files/ftp_upload/63476/63476fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63476/63476fig02.jpg" /> 
Figure 2: Representative sagittal serial sections of the brainstem. (A-C) Left: slices from lateral to medial axis&#160;with the auditory nuclei labeled in a white dashed circle. The middle insert shows the same auditory nuclei region in larger view, marked within white dashed circles. (A) White dashed circle in the center of slice highlights the SON; arrow showing auditory nerve fibers and arrowhead showing NA. A dark black spot at right-side tip of slice is an imaging artifact. Regions of the cerebellum can be seen dorsal to the auditory region in both slices A and B in left panel. (B) A sagittal slice whose orientation was changed to the coronal plane (during slicing). The auditory region was identified with blue dye (black arrow) and again sliced in the sagittal plane. (A-C) Middle insert NM and NL region marked under dashed white lines. Right: satellite view shows a: NM and b: NL observed in 60x objective magnification. LF and HF tonotopic gradient in auditory nuclei is shown along rostro-caudal axis. Arrows pointing to the dark area in (C)&#160;show&#160;heavily myelinated NM fibers running across the midline through the medial axis. The fibers connect either side of auditory nuclei. Abbreviations: NM = nucleus magnocellularis; NL = nucleus laminaris; NA = nucleus angularis; SON = superior olivary nucleus; LF = relatively low-frequency neurons; HF = high-frequency neurons; D = dorsal; V = ventral; R = rostral; C = caudal. <a href="https://www.jove.com/files/ftp_upload/63476/63476fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63476/63476fig03.jpg" /> 
Figure 3: Electrophysiological recordings of neuronal response to somatic current injections (&#8722;100 pA to +200 pA, +10 pA increments, 100 ms duration) in current clamp mode. Neurons were selected for recordings in the same slice but at extreme opposite regions of NM. (A,B) Representative neuronal responses in a single coronal slice indicating relative iso-frequency properties with subtle differences. Response properties represent two different MF neurons recorded from the most medial (A) and lateral (B) regions of NM in a coronal slice. (C,D) Representative neuronal recordings from a single sagittal slice. The recordings show a relatively LF NM response (C) and a HF NM response (D), highlighting the substantive differences in tonotopic gradient along within a single sagittal section. Abbreviations: NM = nucleus magnocellularis; LF = relatively low-frequency neurons; MF = mid-frequency neurons; HF = high-frequency neurons. <a href="https://www.jove.com/files/ftp_upload/63476/63476fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63476/63476fig04.jpg" /> 
Figure 4: Representative horizontal serial sections of the brainstem. (A,B) Left: slices along the dorsal to ventral axis, auditory nuclei are marked with white dashed circles. The 8th cranial nerve afferent fibers connect auditory nuclei marked with arrow. The middle insert is a larger view of auditory nuclei region with auditory nuclei marked under white dashed lines NM and NL regions are shown. A clear topological movement of auditory nuclei can be seen in A,B. (A,B)&#160;Right: large satellite view showing a: NM and b: NL. Right insert shows auditory nuclei observed in 60x objective magnification and the curved topological axis from LF to HF along a caudo-lateral to rostral-medial axis. Abbreviations: NM = nucleus magnocellularis; NL = nucleus laminaris; LF = relatively low-frequency neurons; HF = high-frequency neurons; L = lateral; R = rostral; C = caudal. <a href="https://www.jove.com/files/ftp_upload/63476/63476fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63476/63476fig05.jpg" /> 
Figure 5: Representative horizontal/transverse acute angular (45&#176;) serial sections. (A-C) Left: serial sections of the brainstem, auditory nuclei marked in white dashed circle. The middle insert is a larger view of the auditory region. (A) Middle insert shows the largest spread of NM and NL neurons in these slices. (B,C) Middle insert: auditory nuclei marked in white dashed lines show gradual topological change when compared to (A-C). Right: satellite insert showing auditory nuclei a: NM and b: NL in 60x objective magnification. The tonotopic axis from LF to HF regions in NM and NL rotates angularly from lateral to medial slices. Abbreviations: NM = nucleus magnocellularis; NL = nucleus laminaris; LF = relatively low-frequency neurons; HF = high-frequency neurons; V = ventral; R = rostral; D = dorsal; C = caudal. <a href="https://www.jove.com/files/ftp_upload/63476/63476fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video S1: Hyperpolarizing and depolarizing somatic current injections. Response properties from a low-frequency and high-frequency neuron to 100 ms somatic current injections in current clamp mode. Neurons were selected from the same sagittal brainstem slice. Injections range from -100 to +200 pA in steps of +10 pA increments, 100 ms time duration. Action potentials are seen in response to sufficient depolarizing current steps. The video corresponds to the final traces shown in Figure 3C. <a href="https://www.jove.com/files/ftp_upload/63476/SupplementaryVideo 1 3C - Copy.zip" target="_blank">Please click here to download this File.</a>
Supplementary Video S2: Hyperpolarizing and depolarizing somatic current injections. Similar to Supplementary Video S1, this video shows response properties from a low-frequency and high-frequency neuron to 100 ms somatic current injections in current clamp mode. Neurons were selected from the same sagittal brainstem slice. Injections range from -100 to +200 pA in steps of +10 pA increments, 100 ms time duration. Action potentials are seen in response to sufficient depolarizing current steps. The video corresponds to the final traces shown in Figure 3D. <a href="https://www.jove.com/files/ftp_upload/63476/SupplementaryVideo 2 3D - Copy.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits at JoVE.com

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

slicing-embryonic-chicken-auditory-brainstem-to-evaluate-tonotopic

Research

JoVE Journal

Neuroscience

2.1K Views.  Northwestern University. Deze methode verwerft levende plakjes van de auditieve hersenstam van de kip die een grotere gradiënt van frequentiegebied in hetzelfde segment omvat. Deze plakjes zijn geschikt voor elektrofysiologische en anatomische experimenten met een grotere tonotopische as. Deze methodologie zal helpen om de ontwikkeling van microcircuits in de auditieve hersenstam te begrijpen.Hoewel de anatomie specifiek is voor de kip, hebben andere vogelsoorten zoals kwartels en zangvogels een vergelijkbare...

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

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. ...

Developmental Biology

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 ...

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 ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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.

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 ...

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

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 ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://app.jove.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

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

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

Samenvatting

Meer video's verkennen