Figure 2.

Juxtasynaptic stimulation of identified L5B giant terminals in acute brain slice. A, Experimental layout. Fluorescently labeled terminal and neuron filled with Alexa 594 via patch pipette. A theta glass stimulation electrode producing a very focal electrical field was positioned within 2 μm of the synapse. See Materials and Methods for additional details. B, Simultaneous visualization of slice morphology and synaptophysin-GFP-labeled giant terminals using 2P-SGC microscopy (Wimmer et al., 2004). C, The 2P-SGC image illustrates the recording configuration with the postsynaptic recording electrode (top) and the high impedance double-barrel stimulation pipette (bottom, asterisk). D, As in C, but with green and red fluorescence, by simultaneous online overlay of three channels depicting slice morphology (gray), postsynaptic fluorescence (red), and presynaptic fluorescence (green). The inset shows a higher magnification of the giant terminal and cognate dendrite. E, Current recording from a POm neuron while gradually increasing stimulus intensity. Current traces represent the response before and after reaching the threshold for presynaptic AP. Clamping the voltage at −70 mV caused a small steady-state outward current because of the resting potential of approximately −73 mV. F, All-or-none property of EPSC response. The EPSC peak amplitude as a function of stimulus intensity showed all-or-none behavior. After reaching threshold, the response was independent of stimulation intensity, suggesting that presynaptic APs evoked unitary EPSCs. Relative stimulation intensity was inferred from the amplitude of the stimulus artifact. Scale bars: B–D, 10 μm.