Culturing Hippocampal and Cortical Neurons

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Abstract

Primary cultures of rat and mouse hippocampus and cerebral cortex are widely used to study neuronal properties such as axonal extension, synaptic transmission, and excitotoxicity. Short-term culturing of these neurons can be very straightforward and is perhaps easier than culturing cell lines once dissections are made and cell stocks are frozen. Long-term cultures of relatively pure neuronal populations require slightly more effort, but protocols are described that are less complicated than most published protocols. These include simpler ways to clean and coat coverslips, as well as using glia-conditioned medium to eliminate the need to make individual cocultures of neurons and glia. These methods consistently yield hippocampal and cortical cultures expressing dendritic spines and synapses that survive over 3 weeks in culture. For investigators employing biochemical assays where a fairly large amount of protein is necessary, cortical neurons may be especially attractive to use as large amounts of tissue are obtained and available for culture.

Introduction

Cultured neurons from rodent hippocampus and cerebral cortex have been popular for studying a variety of cell functions. Short-term cultures of these neurons are useful for studying neurite initiation and extension, as well as the establishment of cell polarity. The ability of these neurons to survive for weeks in culture and form synapses allows for studies of synaptogenesis and synaptic transmission. At all stages these neurons have been used for studies of mRNA and protein trafficking and targeting, signal transduction, and excitotoxicity. Part of the popularity of these cultures stems from their physiological relevance. Both the cortex and the hippocampus are primary sites for acquisition and storage of memories, and are regions significantly affected by neurodegenerative diseases such as Alzheimer's disease, epilepsy, and stroke. The corticospinal tract is also damaged after spinal cord injury, for example. These properties make these cultures excellent and accessible models for studies of normal neuronal development, as well as neurodegeneration.

Hippocampal and cortical neurons are also good models because the synapses formed in culture are not completely unnatural. In the intact hippocampus, pyramidal cells send out recurrent collaterals, which form synapses on other pyramidal cells. In the intact cerebral cortex, a large proportion of cortical neurons also normally synapse on cortical neurons in other layers of the cortex.

The majority of cells in hippocampal cultures obtained from embryonic rats or mice are pyramidal neurons having a highly characteristic morphology, plus a smaller component of interneurons and glia. A drawback of these cultures is the relatively small amount of tissue obtained from hippocampal dissections. A much greater amount of tissue is available from cortical cultures, which is important if doing biochemical assays or Western blots. A drawback of cortical cultures is the greater cell heterogeneity, but there still remain a large fraction of neurons having classical pyramidal neuron morphology.

In general, short-term cultures (<5 days) do well grown in a variety of media, either with serum or in serum-free medium with growth supplements. However, because longer term cultures lose viability, give inconsistent results, or become overrun with glia if grown in this manner, studies requiring long-term cultures and synapse formation have used more complex culture methods that involve the presence of differentiated glia. The most commonly used method is the classic “Banker method” (Goslin et al., 1998), which involves growing hippocampal neurons on coverslips placed in close apposition to a bed of glia. The Banker method can give beautiful cultures and allows for the harvest of fairly pure neuronal cultures, but is a relatively complicated method. Another commonly used method for long-term cultures is to grow a confluent culture of glia and then plate neurons directly on the glial bed. This method has been useful for electrophysiological studies (e.g., Bekkers and Stevens, 1989) and studying individual neurons. However, glia contamination prevents biochemical analyses of neurons alone.

Our goal is to describe methods for culturing hippocampal and cortical neurons that are relatively simple, but which give consistent, healthy cultures. The first method described is useful for cultures grown for a few days. The second method, using glia-conditioned medium, is useful for longer term cultures grown more than 10 days, when synapses form. These methods are modifications of culture methods described previously by several laboratories (Mattson 1988, Brewer 1993, Goslin 1998). We have used the short-term culture methods for rat hippocampal and cortical neurons to study signal transduction⧸phosphorylation (Meberg et al., 1998), effects of adenovirus-mediated overexpression of ADF⧸cofilin on neurite outgrowth (Meberg and Bamburg, 2000), and effects of neurodegenerative stimuli on actin inclusion body formation (Minamide et al., 2000). We are currently using the long-term cultures to characterize the function of ADF⧸cofilin at synaptic sites. Protocols are described in detail for rat neurons, but these methods should work equally well for mice, as many laboratories have published studies using similar methods for mouse hippocampal and cortical neurons (e.g., Xiang 1996, Ferreira 2000, Hasbani 2001).

Section snippets

Acquisition of Hippocampal and Cortical Neurons

Both cortical and hippocampal neurons can be obtained from the dissection of a single pregnant rat. Preparation, dissections, cell dissociation, and freezing of cell stocks can be completed in less than 3 h. With this relatively minimal time investment you can have cells ready for experiments for several weeks to follow, especially if using cortical tissue. Dissections are not difficult after minimal practice, if dissection tools are of good quality and a decent dissecting microscope is

Plating of Neurons

For studies on neurons grown less than 10 days in culture, culture methods are simple and cells survive well by plating in Neurobasal⧸FBS and then switching the medium to neurobasal containing B27 supplements (Table III). Because we typically use these cultures within 4 days, no feeding is necessary. Otherwise they should be fed by replacing one-half of the medium every 4–7 days. Cells are plated on cleaned coverslips (Table IV) or plastic dishes, which are then coated with poly-d-lysine (Table

Long-Term Culture Methods

We wanted to have neurons in culture that consistently formed active synapses and spines without contaminating glia so that we could readily visualize dendritic arbors and do biochemical assays with neuronal protein, not glial protein. We also did not want the time commitment and complication involved in the many steps of the Banker method and wanted to be able to plate many neurons on 35- or 60-mm tissue culture dishes. To accomplish this, neurons are initially plated as described for

Summary

We have described protocols for the short- and long-term culture of healthy hippocampal and cortical neurons. The protocols for cleaning and coating coverslips or culture dishes are faster and simpler than many commonly used protocols. The use of frozen stocks of neurons and glia also greatly reduces the frequency of required dissections. Finally, the use of glia-conditioned medium is (1) effective in producing long-lived neurons with spines and synapses and (2) easier and less complicated than

Acknowledgements

Thanks to Steve Schmidt and Kelsey Thibert for performing assays on glia number, neurite lengths, and cell survivability. This work was supported in part by the National Institutes of Health (NS40760) and a Basil O'Connor Starter Scholar Research Award (5-FY99-850) from the March of Dimes Birth Defects Foundation.

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