Preparation and Culture of Chicken Auditory Brainstem Slices

Summary

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.

Abstract

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.

Protocol

1. Preparation of Dissecting Area

1. Continuously bubble artificial cerebral spinal fluid (ACSF) with a mixture of 95% O₂ / 5% CO₂ (pH between 7.2-7.4, osmolarity 295-310 mOsm/l).
2. While ACSF is bubbling, clean working area with 70% EtOH. Also clean the vibratome and slicing blade. Rinse blade with distilled water (dH₂O).
3. Place clean fluid absorption pad on dissecting area with appropriate dissecting tools.
4. Glue agar block (40 mg agarose / mL, i.e. 4%, in dH₂O) to the stage of a vibratome-slicing chamber*.
   *Tip: prepare agar prior to dissection. Store pre-made agar inside a covered petr....

Discussion

For several decades, the acute slice preparation of the chicken brainstem has been used to study auditory processing ^{9, 10}. This approach has provided a tremendous amount of in-vitro physiological data on binaural processing from both developmental and mature states ^{4, 11, 12}. Much is known about this highly specialized circuit and the role each nucleus plays in the temporal processing of sound ^{13, 14}. In fact, short-term experimental manipulations and their effects on the struct.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chicken ACSF
NaCl		Fisher Scientific	M-11624	130 mM
NaHCO3		Fisher Scientific	M-10576	26 mM
KCl		Sigma-Aldrich	P-9333	3 mM
NaH2PO4		Sigma-Aldrich	S-8282	1.25 mM
Glucose		Sigma-Aldrich	G-7528	10 mM
MgCl2		Fisher Scientific	M33-500	1 mM
CaCl2		Acros Organics	423525000	2 mM
Chicken culture medium (store at 4°C)
advanced minimum essential medium (with NEAA, sodium pyruvate at 110 mg/l, without L-glutamate)		GIBCO, by Life Technologies	12492-013	48.00%
Earl’s balanced salt solution		Sigma-Aldrich	E-2888	24.00%
L-glutamine (200 mM)		Sigma-Aldrich	G-7513	1.00%
Glucose solution (200 g/l, sterile filtered)		Sigma-Aldrich	G-7528	2.75%
horse serum (heat inactivated, sterile filtered)		Sigma-Aldrich	H-1138	24.00%
Penicillin-streptomycin*		Sigma-Aldrich	P-0781	1.00 ml * = Add to 100 ml culture medium if needed to prevent contamination
Specific equipment
Millicell-CM cell culture inserts	PICMORG50	EMD Millipore
6-well plate	6 Well Cell Culture Cluster	Corning
Incubator	Forma Scientific CO2 Water Jacketed Incubator	Forma Scientific