Neuronal differentiation of the early embryonic auditory hindbrain of the chicken in primary culture

Abstract

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.

Identification of auditory neurons by retrograde labelling for patch-clamp recordings in a mixed culture of chick brainstem

“…The expression starts between embryonic day 6.5 (Kuenzel et al, 2007) and day 11 (Parks et al, 1997) for NM neurons and 6.5 and 13 for NL neurons. Calretinin expression seems to be independent of auditory input (Parks et al, 1997) and was previously used to identify auditory brainstem neurons in vitro in a mixed culture preparation of a much earlier explantation age (E6.5; Kuenzel et al, 2007). We find a comparable increase in the percentage of calretinin expressing neurons from 36% at DIV 1 to 45% at DIV 6 (35% at DIV 0 and 44% at DIV 7; Kuenzel et al, 2007).…”

“…Calretinin expression is a faithful marker for auditory brainstem neurons (Parks et al, 1997;Kuenzel et al, 2007). The expression starts between embryonic day 6.5 (Kuenzel et al, 2007) and day 11 (Parks et al, 1997) for NM neurons and 6.5 and 13 for NL neurons.…”

“…Calretinin expression is a faithful marker for auditory brainstem neurons (Parks et al, 1997;Kuenzel et al, 2007). The expression starts between embryonic day 6.5 (Kuenzel et al, 2007) and day 11 (Parks et al, 1997) for NM neurons and 6.5 and 13 for NL neurons. Calretinin expression seems to be independent of auditory input (Parks et al, 1997) and was previously used to identify auditory brainstem neurons in vitro in a mixed culture preparation of a much earlier explantation age (E6.5; Kuenzel et al, 2007).…”

“…Calretinin expression seems to be independent of auditory input (Parks et al, 1997) and was previously used to identify auditory brainstem neurons in vitro in a mixed culture preparation of a much earlier explantation age (E6.5; Kuenzel et al, 2007). We find a comparable increase in the percentage of calretinin expressing neurons from 36% at DIV 1 to 45% at DIV 6 (35% at DIV 0 and 44% at DIV 7; Kuenzel et al, 2007). This increase could be either due to quantitative up-regulation of calretinin expression in auditory neurons from expression levels initially below detection level or a differential survival of auditory vs. non-auditory neurons in the culture.…”

“…Recently we published a study on the development of a mixed culture of chicken auditory brainstem neurons (Kuenzel et al, 2007). We could show that these neurons express calretinin and characteristic voltage-gated potassium channels Kv3.1b in vitro, and we could also identify different firing types by whole-cell patch recordings.…”