Optimization of serum-free culture conditions for growth of embryonic rat cholinergic basal forebrain neurons

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Abstract

The objective of the present study was to optimize conditions for culturing embryonic rat basal forebrain neurons in serum-free defined medium to be used in investigations of cholinergic neuron function and responsiveness to neurotrophic factors. It was determined that a combination of neurobasal medium (NB) and DMEM/F12 medium (DM:F12) maintained culture viability, basal choline acetyltransferase (ChAT) activity and responsiveness of these neurons to nerve growth factor (NGF) better than growth of neurons in either medium alone; all media tested contained N2 supplements. While NB which was developed initially for culturing embryonic rat hippocampal neurons supported the growth of basal forebrain neurons, they had reduced ChAT activity and did not respond to NGF with enhanced cholinergic neuronal enzyme activity. On the other hand, DM:F12 did not consistently support survival of the neurons until assay of ChAT activity on day 6 in vitro; surviving cultures were compromised in their cholinergic capacity either under basal or NGF-enhanced conditions. Cultures grown in the combined media responded to brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor (CNTF), at concentrations up to 100 ng/ml with increased ChAT activity as predicted from the literature. These findings suggest that the nutrient composition of the medium is important in promoting expression of the cholinergic neuronal phenotype and that growth factor supplementation alone is insufficient to compensate for inadequate nutrient composition.

Introduction

Growth of neurons in primary culture under serum-free conditions has become more common since defined supplements were developed to replace the use of undefined biological fluids in media (Bottenstein and Sato, 1976Bottenstein, 1983Bottenstein, 1984). These cultures are useful for studying the effects of growth and differentiation factors on neuronal signalling mechanisms for several reasons. Firstly, relatively pure cultures of neurons can be obtained minimizing non-neuronal cell types such as glia, thereby facilitating study of more direct influences of these factors on neurons. Secondly, serum-free media provide precisely defined experimental conditions by eliminating unknown growth and differentiation factors and toxins found in serum which could mask responses of cultured cells to test factors; this latter effect is important as many growth factors act within narrow concentration ranges. Thirdly, undefined serum components in the culture system can alter normal cell development, promote inappropriate differentiation and growth of the target neuron population, or alter gene expression. In addition, batch to batch variability in the composition of sera reduces reproducibility of culture conditions and biological responses.

Success of an experimental paradigm depends on establishing appropriate culture conditions for the biological phenomena being studied. For primary neuronal cultures, the first consideration is providing optimum conditions for neuron survival. Neurons from different brain regions may show variable survival rates under given culture conditions. Brewer (1995)observed that survival of primary cultures of neurons from cortex, septum, substantia nigra, striatum and hippocampus of embryonic rat brain differed when maintained in media of identical formulation. Culture conditions must also be chosen which support the basal physiological function of the neurons and allow expression of the neurotransmitter phenotype characteristic of the sub-population of neurons. To be an effective model system for investigations of effects of growth factors or other agents on neurons, culture conditions must support expression of appropriate cell surface receptors if the events being studied are to approximate physiological events.

This paper describes a serum-free culture system optimized for growth of cholinergic neurons of embryonic rat basal forebrain. A variety of media formulations were tested with culture viability until assay and expression of the cholinergic neuronal phenotype as determined by specific activity of choline acetyltransferase (ChAT) being monitored. In addition, as a measure of physiological responsiveness of the cholinergic neurons, nerve growth factor (NGF)-mediated changes in ChAT activity was monitored as it has been demonstrated previously in both in vivo and in vitro studies that NGF enhances specific activity of this cholinergic phenotypic marker (Hefti et al., 1985Hayashi and Patel, 1987Takai et al., 1988Rylett et al., 1993Pongrac and Rylett, 1996).

Section snippets

Isolation of embryonic neurons

Basal forebrain regions were dissected from embryonic Sprague–Dawley rats at gestational day 17 according to the method of Hefti et al. (1989). Individual cells were isolated mechanically by trituration of minced tissue in Hanks Ca2+- and Mg2+-free Balanced Salt Solution (HBSS) supplemented with 10% fetal bovine serum (FBS; Hyclone) with 9-inch fire-polished Pasteur pipettes of successively smaller bore size. After dispersion, cells were filtered through a 70 μm filter (Falcon) into a 50 ml

Results

Neuronal cultures from embryonic rat basal forebrain were grown in defined media of various compositions based on combinations of NB and DM:F12 with the addition of N2 medium supplements. To produce relatively pure neuronal cultures, a selective plating procedure was used to reduce the number of non-neuronal cells present at the outset of culture, and growth of glial cells and other non-neuronal cells was inhibited by the absence of serum in the culture medium. Cultures were maintained for up

Discussion

In the present study, comparisons were made between the ability of different serum-free media formulations to maintain primary cultures of embryonic basal forebrain neurons and support cholinergic neuron responsiveness to neurotrophic factors. Two commercially available media were used alone or combined in various ratios; all media contained the N2 supplements (Bottenstein and Sato, 1976, Bottenstein, 1983, Bottenstein, 1984) at concentrations used for culture of medial septal neurons (Mazzoni

Acknowledgements

This work was supported by a grant to RJ Rylett and a studentship to JL Pongrac from the Ontario Mental Health Foundation. The authours thank Jacqui Baskey for technical assistance, Dr MJ Strong for provision of the neurofilament antibody, Dr L Weaver for provision of the CNTF, and Regeneron for provision of the BDNF.

References (38)

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