Specific Culture Methods

CNS neurons can be cultured in a variety of formats, depending on the desired experimental paradigm. For most physiologic experiments, neurons are cocultured with glial cells in a relatively enriched medium (Dichter, 1978). For other experiments neurons can be grown in an almost glial-free environment with carefully controlled and characterized media (Brewer, 1995; Brewer et al., 1993; Evans et al., 1998). Cells can be grown at varying densities, depending on the nature of the experiment to be performed. For example, for routine physiology and pharmacology, neurons are often grown at relatively high density on top of proliferating astrocytes. These neurons are best studied at a density that allows identification of individual cells (soma and dendrites) in a microscopic field filled with many neurons. Under these conditions the neurons are healthy, synapse formation is abundant, and small networks develop (Dichter, 1978). For biochemical experiments, cultures such as these can be grown on the surface of large plates or in flasks.

One drawback of the usual "high-density" cultures is that finding pairs of synaptically connected neurons is difficult. In these cultures, all the neurons receive abundant excitatory and inhibitory synaptic inputs, but when one records from pairs of neurons near one another, connections are rarely found. By growing cultures at very low densities, such that only two or three neurons are visible in a microscopic field, coupled pairs are easily found (Wilcox and Dichter, 1994; Wilcox et al., 1994). An extreme example of this arrangement is found when neurons are cultured in isolation, often seeded at very low densities onto astrocytes or microdots of adherent substrate (Segal, 1991; Segal and Furshpan, 1990). Under these conditions, neurons form abundant autapses onto themselves such that each neuron is both presynaptic and postsynaptic to itself. When the neuron is glutamater-gic, receptor antagonists must be included in the medium to prevent excitotoxic autostimulation.

There are many subtle variations on the main method-ologic scheme employed to establish CNS cell cultures. These can best be obtained from either original papers or from texts devoted to CNS cell cultures (Buchhalter and Dichter, 1991; Dichter, 1978; Wilcox et al., 1994). In general, pregnant rats (or mice) are anesthetized, fetuses are removed, and hippocampus or neocortex is dissected (hippocampus at 18 to 21 days of gestational age, neocortex at 15 to 21 days). The brain tissue is dissociated by incubation in trypsin, minced into smaller pieces, and triturated by drawing the tissue back and forth through a pipette tip. Some investigators prefer to avoid enzymes, and dissociate the cells by mechanical methods alone. This latter procedure tends to produce a much larger number of cells, but with a much lower viability. Particulate matter settles out of the mixture; remaining single cells are suspended in media, counted, and plated onto special glass coverslips or plastic tissue culture plates at specific densities. Both the coverslips and plates are coated with an adherent substrate, most often polylysine or polyornithine. Collagen can also be utilized, but neurons grown on collagen have more of a tendency to develop in clumps and remain less dispersed. For routine cultures, cells are plated at 400 to 600/mm in media containing 5% or 10% serum. A variety of enriched growth media can be employed (Banker, 2002). For very-low-density cultures, cells are plated at 100 to 110/mm2 in medium containing high (20mM) potassium concentration. Cultures remain in CO2 incubators at 37° C until they are used.

Figure 1 illustrates sample microscopic fields from hip-pocampal cultures grown under three conditions: high density (400,000 cells/35-mm plate) with serum; low density (60,000 cells/35-mm plate) with serum and high K; and intermediate density (100,000 cells/35-mm plate) in neurobasal medium without serum. In the phase-contrast images of the top row, one can see astrocytes underlying the neurons at 2 and 3 weeks. These cells are absent or very sparse in the neurobasal cultures.

Within hours of plating, most viable cells settle on the substrate, adhere, and begin sending out processes. Even at this early point, some neurons appear pyramidal, some stellate, and some bipolar. Whether these morphologies correspond to what the cells were destined to become in vivo has never been determined. By 3 to 5 days in vitro (DIV), the neurons have clearly differentiated and have developed extensive processes. Depending on the brain region being cultured and the age at which it was dissected, synapses begin to appear at about 4 to 7 DIV and increase in density and complexity for days thereafter. Given the nature of the connectivity in the high density cultures, it is likely that axons extend fairly significant distances before synapses are made. Cultures of this kind can be maintained in a robust state for 4 to 6 weeks or longer. As the cultures age, there appears to be a gradual decline in the number of viable neurons, although those that remain often are quite large and have extensive dendritic trees and abundant spines.

When cultures are grown in high density with astrocytes, it is often necessary to use mitotic inhibitors (for example cytosine arabinoside at 10 ||M) at about 4 to 7 DIV to prevent overgrowth of glia and loss of neurons. This procedure does not appear to affect the neurons adversely. High-density cultures grown in defined media have minimal astrocytes and do not require mitotic inhibition. Despite the lack of astrocytes in low-density cultures, the neurons appear quite normal and form abundant excitatory and inhibitory synapses.

When synaptic interactions are to be studied using paired recordings, cultures are often grown at very low density. These cultures are maintained in lower volumes of media containing higher than usual amounts of potassium (e.g., 20mM) (Mattson and Kater, 1988, 1989). Under these conditions, glial cells grow very slowly and do not overgrow the cultures (Wilcox et al., 1994). The basic properties of these neurons appear similar or identical to those grown at higher density, although this similarity has not been rigorously studied.

FIGURE 1 Three sets of hippocampal dissociated cell cultures: at 1 day (left column), 2 weeks (middle column), and 3 weeks (right column) in vitro. For each field, there is a photomicrograph of the same field (40x magnification) in phase contrast (left) and after neuronal staining with antibodies to MAP2 (right). The top row shows cultures grown at high density (400,000 cells/35-mm plate) with serum; the middle row shows cultures grown at low density (60,000 cells/35-mm plate) with serum and high K; the bottom row shows cultures grown at intermediate density (100,000 cells/35-mm plate) in neurobasal medium without serum. In the phase-contrast images of the top and middle row, one can see astrocytes at 2 and 3 weeks. These cells are absent or very sparse in the neurobasal cultures.

FIGURE 1 Three sets of hippocampal dissociated cell cultures: at 1 day (left column), 2 weeks (middle column), and 3 weeks (right column) in vitro. For each field, there is a photomicrograph of the same field (40x magnification) in phase contrast (left) and after neuronal staining with antibodies to MAP2 (right). The top row shows cultures grown at high density (400,000 cells/35-mm plate) with serum; the middle row shows cultures grown at low density (60,000 cells/35-mm plate) with serum and high K; the bottom row shows cultures grown at intermediate density (100,000 cells/35-mm plate) in neurobasal medium without serum. In the phase-contrast images of the top and middle row, one can see astrocytes at 2 and 3 weeks. These cells are absent or very sparse in the neurobasal cultures.

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