(A) In trace eyeblink conditioning, animals associated a neutral conditioned stimulus (CS) with electrical shock near the eyelid (US) over a temporal gap (500 ms). With repeated pairings, rats developed anticipatory blinking responses before US onset (CRs), which were monitored by recording electromyogram (EMG) from the upper eyelid (a.u., arbitrary unit). (B) Daily conditioning took place in a conditioning chamber which consisted of two visually distinct rooms (Box 1 and 2) connected by a short walkway. (C) Rats first entered Box 1 and received an auditory stimulus (ACS) alone for 20 trials (CS-alone block), followed by 50 ACS-US pairings (CS-US block). They then moved to Box 2, where they received 20 ACS alone trials followed by 50 ACS-US paired trials. Finally, they returned to Box 1 and received a visual stimulus (VCS) alone for 20 trials followed by pairings of the VCS and US for 50 trials. (D) The frequency of CR expression increased in the CS-US paired, but not in the CS-alone, trials (mean CR% ± SEM; n = 7 rats; Sessions × Trial blocks interaction, F75, 450 = 2.992, p<0.001). H1 and H2 show the rate of blinking when only the CS was presented before any conditioning. (E) In the last session, CR% (in every 10 trials, mean ± SEM; n = 5 rats that received the CS-alone block before the CS-US paired blocks) showed an abrupt transition upon the shift from the block of CS-alone trials (light colors) to the block of CS-US paired trials (dark colors). (F) A trial-by-trial pattern of CR expression of Rat 2 during the last session. (G) The proportion of rats (n = 4 rats underwent the trial blocks in the same temporal sequence) that showed the CR in each trial during the last session.