Figure 1. Properties of reciprocal inhibition.

A, quantification of reciprocal inhibition. An axotomized terminal was depolarized from −70 to −10 mV for 200 ms. The charge of reciprocal IPSC (reciprocal Q_IPSC) was calculated by integrating the difference between the averaged current trace (black) of three responses (grey) and the peak normalized I_Ca template (broken line; see also Fig. S1A). The I_Ca template was obtained as the average of responses recorded from 11 axotomized terminals which were depolarized from −70 to −10 mV for 200 ms in the presence of bath-applied 200 μm picrotoxin and 10 μm strychnine. Twenty mm Hepes was included in the bath solution to suppress the proton feedback on I_Ca (Palmer et al. 2003). The equilibrium potential for Cl⁻ (E_Cl) was −55 mV. The inset shows the expanded traces. B, voltage dependence of Q_Ca (light grey; n= 4–11), ΔC_m (dark grey; n= 4–7), and reciprocal inhibition (black; n= 4–11). Axotomized terminals were depolarized from −70 mV to various membrane potentials (V_m) for 200 ms (Fig. S1B). Q_Ca was the charge of the current response during the 200 ms pulse in the presence of puff-applied BIC and TPMPA. Capacitance jump ΔC_m was determined as the difference between the averaged capacitance before the stimulus onset (from −220 to −20 ms) and that after the stimulus offset (from 500 to 700 ms). The amount of reciprocal inhibition was quantified as Q_IPSC/(V_m−E_Cl), where Q_IPSC is the charge of the reciprocal IPSC obtained as the difference between the current responses before and during puff application of 100 μm BIC and 800 μm TPMPA. Data are shown as the ratio to the value at −10 mV. E_Cl was −70 mV. C and D, effects of various pharmacological blockers on the reciprocal Q_IPSC obtained as in A. The asterisk indicates significant difference between the control and each blocker (+) condition by Welch's t test with Bonferroni correction. E, glutamate (5 mm) was puff applied for 10 ms (arrowhead) to an axotomized terminal voltage clamped at −10 mV in the absence (black) or presence of bath-applied 200 μm Cd²⁺ (grey). F, the glutamate-evoked Q_IPSC/800 ms (see Methods) became smaller as the puff pipette was laterally shifted from the recorded terminal (n= 10). Data were fitted to a single exponential function and the length constant (λ) was 8.5 μm. BIC, bicuculline; d-AP5, d-(–)-2-amino-5-phosphonopentanoic acid; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide; PhTX, philanthotoxin-433; TPMPA, (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid; TTX, tetrodotoxin.