Astrocyte-conditioned saline supports embryonic rat hippocampal neuron differentiation in short-term cultures

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Abstract

Embryonic rat hippocampal neurons were cultured for 1–2 days in serum-free, HEPES-buffered Tyrode's solution. The effects of cortical astrocytes and astrocyte-conditioned saline on neuron survival, membrane surface area and the expression of functional amino acid neurotransmitter receptors were studied. Neurons grown in Tyrode's solution alone survived well for 1 day but deteriorated thereafter both in terms of percent neurons surviving and the amplitudes and densities of GABA-, glycine-, kainate- and NMDA-induced currents. Neurons grown in Tyrode's previously conditioned by astrocytes for 24 h had significantly larger apparent plasma membrane surface area, as indexed by whole-cell membrane capacitance, and larger amplitudes and densities of the amino acid-induced currents after both 1 and 2 days. The survival rate and neurite outgrowth were also greater in the astrocyte-conditioned saline group after 2 days in culture. Similarly, neurons cultured on glass cover-slips facing a confluent monolayer of astrocyte were larger in apparent plasma membrane area and amino acid-induced currents than neurons cultured in Tyrode's alone. Neurons cultured in saline conditioned by astrocytes provide a strategy to study the physiological basis of astrocyte-directed neuronal differentiation in the absence of ambiguities arising from the inclusion of sera and other additives often used in vitro.

Introduction

Cultured embryonic mammalian neurons and glia have been widely used to reduce the cellular complexity inherent in the intact CNS and to study developmentally changing properties in detail (Banker and Goslin, 1991). However, some components contained in commonly used culture media, like fetal calf and/or horse serum, may either by themselves exert ambiguous effects on developmentally-relevant properties or could obscure or interfere with the biologically active substances to be tested. Although horse serum and/or other complex components are often used, in an empirical manner, to sustain long-term (weeks) cultures of developing neurons, rapid changes in neuronal properties that occur within hours–days of plating (e.g. Liu et al., 1996) may not require either serum-containing or defined medium enriched with additives. For example, we found significant differences between GABAA receptor/Cl channel functions expressed by neurons plated on poly-d-lysine and those on a confluent monolayer of astrocytes (or in astrocyte-conditioned medium) within 2 h (Liu et al., 1996). These and other differences in neuronal morphology and excitability either were maintained or became further evident during 24–48 h (Liu et al., 1996, Liu et al., 1997). These effects of astrocytes on embryonic hippocampal neuron differentiation may be physiologically relevant since astrocytes emerge at the end of embryogenesis in the hippocampus. Here, we report a simple method to culture embryonic rat hippocampal neurons in a physiological saline (HEPES-buffered Tyrode's solution) either conditioned by astrocytes or with co-cultured astrocytes that supports the early phase of neuronal development and differentiation with considerable fidelity compared to serum-containing or defined media. This strategy should be useful to investigate the physiological effects of soluble signals released from astrocytes that contribute to neuronal differentiation.

Section snippets

Culture of rat cortical and hippocampal astrocytes and collection of conditioned medium

Monolayers of cortical and hippocampal astrocytes were prepared as described previously (Liu et al., 1996). Cortices or hippocampi were removed from 3-day-old rat pups, cleaned of meninges and placed in 10 ml L-15 medium containing 50 U/ml gentamicin. The tissues were mechanically dissociated and passed through a 70 μm Falcon nylon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ). The dissociated, single-cell suspension was centrifuged at low speed (∼80×g) for 15 min and resuspended

Embryonic rat hippocampal neurons can be cultured for a short-term in HEPES-buffered Tyrode's solution

In an attempt to simplify the culture medium, we grew rat hippocampal neurons isolated from E19 embryos in HEPES-buffered Tyrode's solution or in Tyrode's previously conditioned for 24 h by rat cortical or hippocampal astrocytes. Virtually all of the cells surviving in Tyrode's solution were TUJ1-positive and thus neuronal. Some of the neurons had long, branched processes while others had short, unbranched processes after 1 day in culture (Fig. 1a,b). The density of viable neurons was not

Development of the method of short-term culturing of embryonic rat hippocampal neurons in simple saline

For long-term culture of mammalian neurons, horse serum and fetal calf serum are often used in combination with complex media composed of a variety of inorganic salts, amino acids, vitamins and other components. It is of no surprise that different results are frequently obtained when different batches of sera are used due to their complex and undefined components. Furthermore, proteins and other components in sera may interfere with the effects of diffusible substances (like those released by

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