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Acute isolation and culture of nerve cell from the cotton bollworm and the patch-clamp study on the voltage-gated ion channels in the cultured neurons

The dissociation and culture of cells isolated from the ventral nerve cord of the larva of the cotton bollworm, Helicoverpa armigera are described. The propterties of the voltage-gated ion channels of the cultured neurons were studied with patch-clamp technique in the whole-cell configuration. Neurons survived and grew fast in either TC-100, Leibovitz's L-15 or Grace medium supplemented with glutathione, glutamine, glucose and 10% foetal calf serum. The medium made up of equal parts of TC-100 and Leibovitz's L-15 enabled better adherence, survival and growth. Most neurons showed neurite growth exhibiting unipolar, bipolar or multipolar. Neural cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell patch-clamp recordings. Depolarization of the cell under voltage-clamp resulted in TTX-sensitive sodium currents, high-voltage activated calcium currents and two types of outward potassium currents. The potassium currents could be decreased by TEA-Cl and 4-AP.