Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties

Abstract

1. Reliable methods for establishing fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) cells in long term (greater than 1 mo) dissociated cell cultures are described. These cells have been studied by morphologic and intracellular electrophysiologic techniques. 2. Cells studied electrophysiologically can be relocated after preparation for electron microscopy and examined in thin sections. The electron microscope shows that the surface membranes of these cells were directly accessible to the culture medium. The surfaces of SC cells were studded with synaptic boutons, whereas the DRG cell surfaces generally had none. 3. Current-voltage relationships and linear electrotonic properties of the neurons are described. Delayed and anomalous rectification were seen in both cell types. The length of SC cell dendrites was about one characteristic electrotonic length, while little or no contribution of the relatively sparse DRG cell processes was seen in the transient responses of the DRG cells. 4. Postspike and posttetanic hyperpolarizations in DRG cells were due to a surface membrane conductance increase; this was probably primarily an increase in K+ conductance. Post-activation hyperpolarization in SC cells was primarily due to activation of an electrogenic Na+ pump.

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

“…In addition these results suggest that the increased fluorescence intensity from the sodium channel labeling was not due to localized foldings of the membrane which could enhance the fluorescence intensity by increasing the effective surface area. This is also supported by reports on the fine structure of these cells from other laboratories which have shown that there are no invaginations of the cell membrane around the axon hillock and that the cell surface is topographically uniform over all parts of the cell (42). To confirm that the punctate fluorescence and nonuniform labeling was due to sodium channels rather than the ligands per se, three different sodium channel probes, TmRhd-Lqq V, CPM-Css II (or TmRhd-Tityus y), and NBD-TXX, each specific for a distinct receptor site on the sodium channel protein complex, were used (5).…”

“…In large neurons some of the terminal regions have discernable and identifiable boutons. Other laboratories have shown that these morphologically identifiable connections or appositions are correlated with a high degree of synaptic activity (28,42), In addition, electrophysiological recordings from these regions have shown that the evoked excitatory postsynaptic potentials approximate a normal distribution with no evidence of preferred amplitudes as might occur with relatively low mean quantum content (28,42). Of course at our level of morphology and microscopic resolution we cannot draw any definite conclusions as to the functional properties of each of these terminal structures, but for FPR measurements and for physiological exploration of cells regions, the visualization by phase or differential interference contrast microscopy provides a useful guide.…”

“…Growth of nonneuronal cells was eliminated by adding 5-fluoro-2'-deoxyuridine and cytosine arabinoside to the cultures. Rat embryonic spinal cord cell cultures were prepared using the method of Ransom et al (42), and plated on 0.13-ram-thick glass coverslips coated with polylysine. Cells were maintained in a medium of CO2-equilibrated DME (7% CO2) with 10% fetal calf serum and 10% horse serum.…”

“…Cultures were studied after 2-6 wk of development in vitro at which time functional synaptic connections measured electrophysiologically (28,42), the maturation of neurons, and the densities of sodium channels assessed by neurotoxin binding, were at a maximum. The stage of neuronal growth was determined by immunocytochemistry for the appearance and locale of each of the neurofilament proteins.…”

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells

“…For the preparation of mouse spinal cord primary culture, mouse spinal cords were taken from E12-E13 embryos, dissected and cultured as described by Ransom et al 22 The cords were minced and incubated with 0.1% trypsin in Ca 2 þ Mg 2 þ -free PBS for 15 min, at 371C. Cells were centrifuged (1500 g, 5 min), resuspended and plated on tissue culture dishes or coverslips treated with rat-tail collagen (Jacques-Boy).…”