Minimal stimulation demonstrated the monosynaptic nature of the TS-DMX and TS-dm-cNTS (NTS) transmission. A, IR-DIC microphotograph showing the locations of a theta pipette bipolar electrode (θ) on the TS and recording pipette (p) in the DMX. B, summary of the location of the tip of the theta pipette bipolar electrode and recorded neurons. sfEPSC responses were recorded in the dm-cNTS (n = 7) and DMX (n = 11) neurons. The neurons shown here are not included in Fig. 1D. C, representative traces of sfEPSC in a DMX (left) and a dm-cNTS (right) neuron. Top, superimposed traces of EPSC waveforms that were evoked by minimal paired-pulse stimulation (arrowheads). The stimulation intensity was 75 μA for the DMX neuron and 37 μA for the dm-cNTS neuron. The plots on the bottom show the amplitude of sfEPSC1 and sfEPSC2 while increasing the stimulation intensity from one that evoked only failure responses to one that evoked mostly successful responses with only rare failures. D, success rate (defined as the ratio of non-failure events per trial) of EPSC1 and EPSC2 responses (the left-side ordinate) and PPRsuccess (defined as the success rate of sfEPSC2 normalized by that of sfEPSC1; right-side ordinate) in the 8 DMX (left; open circles) and 7 dm-cNTS (right; filled triangles) neurons. E, histograms (top) showing the distribution of PPRsuccess (filled gray bar; based on the sfEPSC recordings with minimal stimulation) and PPRampli (the PPR estimated based on the amplitude ratio of EPSC1 and EPSC2 with submaximal stimulation; the same histograms shown in Fig. 1C) in DMX (left) and dm-cNTS (right) neurons, respectively. Cumulative probability curves (bottom) produced based on the histograms above. No significant difference was detected between the distributions of PPRampli and PPRsuccess in either DMX (p = 0.95) or dm-cNTS neurons (p = 0.40). Kolmogorov-Smirnov test.