Amino acid pharmacology of mammalian central neurones grown in tissue culture.

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Abstract
Spinal and cerebellar-brainstem areas of fetal mouse were dissociated and grown in tissue culture until large enough to permit stable intracellular recording. The tissue-cultured neurons, growing as a monolayer and accessible under direct vision using phase contrast optics, allowed precise placement of intracellular recording and extracellular ionophoretic pipettes. Ionophoresis of GABA and glutamate revealed a non-uniform distribution of responses over the cell surface, with a lack of spatial coincidence in sensitivity between the two. GABA inhibited and glutamate excited all cells tested. GABA responses evoked at the cell body and on nearby process membrane were almost uniformly hyperpolarizing, while those at some peripheral process membrane were either hyperpolarizing, depolarizing or a combination of both events. All responses were associated with an increase in membrane slope conductance. Membrane polarization showed that all hyperpolarizing events extrapolated to about the same inversion potential, which averaged about 9 m V more negative than resting potential (n = 95). The depolarizing phases of responses evoked at peripheral membranes extrapolated to about 0 mV (n =5). The hyperpolarization and increase in membrane conductance of GABA responses at the cell body were dependent on Cl- and the inversion potential of the response was dependent on the Cl- concentration gradient. The inversion potentials of GABA, glycine and .beta.-alanine responses were identical. When matched in magnitude for evoked conductance increase, glycine responses decayed more rapidly than GABA. Glycine and .beta.-alanine voltage responses both decayed faster than GABA responses of comparable size. In about half the cells tested sustained or rapidly repeated application of GABA and glycine transformed hyperpolarizing responses into depolarizations which were associated with a maintained conductance increase. Results from conditioning-test experiments with pairs of GABA and glycine responses suggested that the reversal of response polarity was due to a rapid redistribution of Cl-. The limiting slope of log-log dose-response curves for GABA-induced conductance averaged about 2, while those for glutamate-induced depolarizations averaged about 1. Molecules (2) of GABA and 1 molecule of glutamate participated in the respective post-synaptic responses. The observations indicate that mammalian CNS tissue grown in culture was a suitable model to study CNS membrane pharmacology with increasing precision.