A primary culture system for biochemical analyses of neuronal proteins
Introduction
Mammalian brain is composed of a variety of cell types, including glial, endothelial, ependymal, and neuronal cells. Even within the neuronal population, there is a wide array of different neuron types (e.g. pyramidal and granule cells, interneurons, glutamatergic, dopaminergic, and cholinergic neurons, etc.). This complexity, together with the poor accessibility of brain tissues, makes difficult the study of protein functions in neurons at the cellular and molecular levels. As such, in vitro cell culture systems have been widely used to investigate molecular events occurring in neurons. A number of neuronal cell culture methods have been established and extensively used to investigate dynamic events leading to the establishment and maintenance of the expression, localization, and function of neuronal proteins. As neurons are non-proliferative, all cultures of mammalian neurons must be of primary origin and limited in number to no more than the neurons originally plated. Moreover, neurons generally require the presence of the critical glial support cells found in the brain, or humoral factors derived thereof, to thrive or even survive, in culture (Goslin et al., 1998).
Mammalian neuronal culture systems can be roughly divided into two major types; high-density cultures of mixed neurons and glia derived from early post-natal brain or low-density cultures of pure neurons derived from embryonic brain and grown in the presence of post-natal astrocytes. Mixed post-natal cultures provide amounts of neuronal protein amenable to biochemical analyses. However, analyses of proteins from these cultures are confounded by the presence of glia and other support cells, especially when the proteins are expressed in neuronal and non-neuronal cells, such as actin-related proteins. The presence of glial cells also complicates pharmacological approaches to dynamic neuronal events because of diverse neuron–glia interactions (Auld and Robitaille, 2003). Pure cultures of embryonic neurons allow for a more precise focus on neuron-specific signaling events and on neuronal proteins. However, the difficulty of obtaining sufficient amounts of neurons for biochemical analysis has hindered many types of studies in the typical low-density pure neuronal cultures. While there have been several recent reports employing biochemical analyses of neuronal proteins in pure neuronal cultures, the procedures employed were not described in detail (Bu et al., 2003, Fujimoto et al., 2004, Ishikawa et al., 2003, Ledesma et al., 2003, Mundigl et al., 1998, Sanchez Martin et al., 2000, Zheng et al., 2002).
In order to study neuronal ion channels in cultured neurons, we developed methods for culturing embryonic rat hippocampal neurons that yields sufficient material for biochemical analyses of neuronal proteins. Here, we report the detailed methods for establishing and maintaining these cultures. We provide a number of examples of neuronal proteins whose expression and biochemical characteristics can be studied in these neuronal cultures. We also show data on the feasibility of using these cultures for analysis of the properties of recombinant proteins expressed in a true neuronal background. Moreover, we provide examples of the feasibility of using dynamic manipulation of these cultures to study activity-dependent changes in the biochemical properties of neuronal proteins.
Section snippets
Materials
All tissue culture reagents were purchased from Invitrogen (Carlsbad, CA) with the exception of animal sera from Hyclone (Logan, UT). Most of other chemicals were obtained from Sigma (St. Louis, MO) and Calbiochem (San Diego, CA) unless otherwise noted. Antibodies used here were described elsewhere (Bekele-Arcuri et al., 1996, Rasband et al., 2002, Trimmer, 1991).
Preparation of culture reagents
We used two types of culture media: plating medium and maintenance medium. The plating medium was modified Eagles medium (MEM)
Results
We have used neurons cultured in the manner described above for analyses of a number of neuronal proteins. Our research has primarily focused on neuronal membrane proteins and associated scaffolding proteins involved in neuronal signaling. Our initial experiments using these cultures focused on applying this culture system to ongoing studies of the voltage-dependent K+ channel Kv2.1, which in mammalian brain is prominently expressed in most neurons (Trimmer, 1991). We had previously obtained
Discussion
Here, we demonstrated that pure cultures of embryonic rat hippocampal neurons could be a useful tool for basic biochemical analyses of neuronal proteins. We provided immunoblot analyses of a variety of neuronal proteins, and also showed that neuronal proteins derived from these cultures can be isolated by immunoprecipitation. We have previously shown that neurons cultured using this procedure can also be used for surface biotinylation assays, and for analysis of post-translational modification
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