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
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Current and former Rubel lab members.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Chicken ACSF | ||||
| NaCl | Fisher Scientific | M-11624 | 130 mM | |
| NaHCO3 | Fisher Scientific | M-10576 | 26 mM | |
| KCl | Sigma | P-9333 | 3 mM | |
| NaH2PO4 | Sigma | S-8282 | 1.25 mM | |
| Glucose | Sigma | 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 Invitrogen | 12492-013 | 48.00% | |
| Earl’s balanced salt solution | Sigma | E-2888 | 24.00% | |
| L-glutamine (200 mM) | Sigma | G-7513 | 1.00% | |
| Glucose solution (200 g/l, sterile filtered) | Sigma | G-7528 | 2.75% | |
| horse serum (heat inactivated, sterile filtered) | Sigma | H-1138 | 24.00% | |
| Penicillin-streptomycin* | Sigma | P-0781 | 1.00 ml * = Add to 100 ml culture medium if needed to prevent contamination |
|
| Specific equipment | ||||
| Millicell-CM cell culture inserts | PICMORG50 | Millipore | ||
| 6-well plate | 6 Well Cell Culture Cluster | Corning Incorporated | ||
| Incubator | Forma Scientific CO2 Water Jacketed Incubator | Forma Scientific |
References
- Howard, M. A., Burger, R. M., Rubel, E. W. A developmental switch to GABAergic inhibition dependent on increases in Kv1-type K+ currents. J Neurosci. 27, 2112-2123 (2007).
- Kuba, H., Koyano, K., Ohmori, H. Development of membrane conductance improves coincidence detection in the nucleus laminaris of the chicken. J Physiol. 540, 529-542 (2002).
- Gao, H., Lu, Y. Early development of intrinsic and synaptic properties of chicken nucleus laminaris neurons. 신경과학. 153, 131-143 (2008).
- Sanchez, J. T., Wang, Y., Rubel, E. W., Barria, A. Development of glutamatergic synaptic transmission in binaural auditory neurons. J Neurophysiol. 104, 1774-1789 (2010).
- Jackson, H., Hackett, J. T., Rubel, E. W. Organization and development of brain stem auditory nuclei in the chick: ontogeny of postsynaptic responses. J Comp Neurol. 210, 80-86 (1982).
- Rubel, E. W., Parks, T. N. Organization and development of brain stem auditory nuclei of the chicken: tonotopic organization of n. magnocellularis and n. laminaris. J Comp Neurol. 164, 411-433 (1975).
- Rubel, E. W., Smith, D. J., Miller, L. C. Organization and development of brain stem auditory nuclei of the chicken: ontogeny of n. magnocellularis and n. laminaris. J Comp Neurol. 166, 469-489 (1976).
- Stoppini, L., Buchs, P. A., Muller, D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. 37, 173-182 (1991).
- Reyes, A. D., Rubel, E. W., Spain, W. J. Membrane properties underlying the firing of neurons in the avian cochlear nucleus. J Neurosci. 14, 5352-5364 (1994).
- Monsivais, P., Rubel, E. W. Accommodation enhances depolarizing inhibition in central neurons. J Neurosci. 21, 7823-7830 (2001).
- Lu, T., Trussell, L. O. Development and elimination of endbulb synapses in the chick cochlear nucleus. J Neurosci. 27, 808-8017 (2007).
- Kuba, H., Yamada, R., Fukui, I., Ohmori, H. Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick. J Neurosci. 25, 1924-1934 (2005).
- Trussell, L. O. Cellular mechanisms for preservation of timing in central auditory pathways. Curr Opin Neurobiol. 7, 487-492 (1997).
- Trussell, L. O. Synaptic mechanisms for coding timing in auditory neurons. Annu Rev Physiol. 61, 477-496 (1999).
- Sorensen, S. A., Rubel, E. W. The level and integrity of synaptic input regulates dendrite structure. J Neurosci. 26, 1539-1550 (2006).
