Unitary, quantal and miniature gaba-activated synaptic chloride currents in cultured neurons from the rat superior colliculus

Abstract

The aim of this study was to identify the conductance change induced by one quantum of γ-aminobutyric acid from axonal release sites on cultured superior colliculus neurons. Unitary (single cell-activated) inhibitory postsynaptic currents and spontaneous synaptic activity were recorded with patch clamp techniques in the whole cell configuration while superfusing the entire neuron with normal saline. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and in reduced[CA 2+] o/[Mg 2+] o. In addition, the membrane area contributing to synaptic activity was limited to a narrow window of 50 μm. Smaller neurons were chosen for recording to render a standard deviation of the “instrumental” noise of less than 1.5pA at a holding voltage of −80 mV. After two weeks in vitro, the percentage of synaptically connected tectal neurons exceeded 50%. At holding voltages of −80 mV (Cl − equilibrium potential −12mV) minimal amplitudes of unitary inhibitory postsynaptic currents were as low as 7–10 pA, while maximal amplitudes exceeded 500 pA. The mean time to peak and time constant of decay were 3.0 and 34.4ms, respectively ( n = 31). Fluctuating unitary inhibitory postsynaptic currents were deemed to be compound postsynaptic responses. Multiple Gaussian equations could be fitted to the amplitude histograms of unitary postsynaptic currents. This procedure rendered a quantal size between 5.0 and 10.9 pA (mean 7.1 pA; S.D. 1.78 pA) in five neurons from mature cultures. The amplitudes of statistically determined quantal inhibitory postsynaptic currents were slightly smaller than the independent estimate from somatic miniature inhibitory postsynaptic currents. The latter had a mean amplitude of 9.1 pA (S.D. 3.3 pA, n = 23), a mean time to peak of 1.65 ms ( n = 9), and a mean time constant of decay of 16.2 ms ( n = 9). Single channel recording from outside-out patches showed three to four main conductance states ranging from 9 to 22 pS. Single channel closures at the 21–24 pS level were occasionally observed during relaxation of miniature currents. The small size of whole cell quantal inhibitory postsynaptic currents and somatic miniature currents indicates that one GABA quantum opened only 5–15 single Cl − channels.