Objective      To investigate whether pseudo-paired presentation of conditioned stimulus (CS) and unconditioned stimulus (US) can facilitate the subsequent CS-US paired training of classical eyeblink conditioning (EBC) in guinea pigs and to analyze the possible neural mechanisms of the facilitation effect.       Methods      Ninety-six health male albino Dunkin-Hartley guinea pigs (4 to 5 months old

weighting 500 to 550 g) were randomly assigned to receive behavioral training of delay eyeblink conditioning (DEBC) or trace eyeblink conditioning (TEBC). Micro-electrical stimulation of the medial prefrontal cortex (mPFC CS) or hippocampus (Hip CS) was selected as the central CS. A pure tone was used as the peripheral CS (Tone CS) and corneal air-puff was applied as US. The animals were divided into 8 experimental groups (n=4) and 8 control groups (n=8). The animals in experimental groups were firstly given 1 week’s pseudo-paired training with central or peripheral CS

and randomly presentation of US

then CS was switched from central to peripheral or vice versa

and paired with US to establish DEBC or TEBC. Animals in control groups were directly given paired training with central or peripheral CS and paired with US.       Results      (1) After the in advance pseudo-paired training with mPFC CS or tone CS and randomly presentations of US

the acquisition rates of both DEBC and TEBC were greater in experimental groups than control groups during paired training in which CS were switched from mPFC stimulation to tone stimulation or vice versa and paired with US (P0.05). (2) After the in advance pseudo-paired training with Hip CS or tone CS and randomly presentations of US

the acquisition rates of TEBC were greater in experimental groups than control groups (P0.01)

but the acquisition rate of DEBC was similar in the experimental groups and control groups (P0.05) when CS was switched from Hip stimulation to tone or vice versa and paired with US.       Conclusion      The early CS-US pseudo-paired training has facilitating effect on the subsequent EBC establishment in CS-US paired training. Two types of non-specific stimulations of both CS and US may contribute to the facilitating effect for EBC in various degrees.